Method for forming a reversible protein nanocluster using light in a cell

ABSTRACT

To efficiently analyze interaction and function between proteins, the present invention relates to a method for forming a light-induced protein nanocluster, comprising: an expression vector preparation step of preparing a first expression vector including polynucleotides coding a first fusion protein including a light-induced heterodimer-forming protein and a first self-assembly protein, and a second expression vector including polynucleotides coding a couple protein that forms a homodimer with said light-induced heterodimer-forming protein, or a second fusion protein including said couple protein and a second self-assembly protein; a transformed cell, tissue or individual preparation step of transforming cells, tissues or individuals using said first expression vector and second expression vector; and a light radiation step of radiating light having a wavelength for inducing the formation of heterodimer between said light-induced heterodimer-forming protein and said couple protein, to said transformed cells, tissue or individuals.

FIELD OF THE INVENTION

The present application claims benefit of priority of Korean PatentApplication Nos. 2011-0100334 and 2011-0130081 filed Sep. 30, 2011 andDec. 7, 2011, respectively. The documents are herein incorporated byreference in its entirety.

The present invention relates to a method for forming a reversibleprotein nanocluster, more particularly to a method for forming areversible protein nanocluster using light in a cell and a method forinhibiting function of a protein reversibly.

BACKGROUND OF THE INVENTION

The dynamic interactions between varieties of biologically activesubstances regulate various physiological functions, and diseases arecaused in an unusual situation when these interactions occur improperlyor the interaction between molecules which should not interact eachother occurs. In general, two proteins having complementary structureinteract and bioactive compounds interact specific parts of tertiarystructure of the proteins. These bioactive compounds may be therapeuticcandidates for diagnosing, preventing, treating or alleviating diseasesinvolved in the proteins by modulating the function of the targetproteins. Studies on screening the disease targets or therapeutics forthe disease have been sustained by analyzing the interaction betweenthese proteins and the interactions between proteins and small-moleculecompounds.

For example, various techniques such as phage display (Sche et al.,Chem. Biol., 6: 707, 1999), yeast two/three-hybrid analysis (Licitra etal., Proc. Natl. Acad. Sci. USA 93: 12817, 1996), and parallel analysisof yeast strain having deletions heterologous (Zheng et al., Chem.Biol., 11: 609, 2004) have been suggested. However, these techniqueshave problems, such as high background, false positive and lowsensitivity of the reaction and with in vitro experiment or reactionsusing non-mammalian cells make it hard to convince the experimentalresult.

In addition, inducing a deletion or a mutation of the gene encoding aprotein or treating specific inhibitor for the protein to the cell orthe subject expressing the protein are used to observe thephysiochemical changes in the cell or the subject.

Among these methods, U.S. Pat. No. 5,270,163 discloses a method forscreening specific biomolecules that bind specifically tosingle-stranded oligonucleotides, the so-called aptamer. Lunder et al.discloses a method of screening target-binding motif using phage display(Lunder et al., Appl. Biochem. Biotechnol., 127 (2): 125-131, 2005).U.S. Patent Publication No. 2010-0143371 discloses a method forinhibiting the function of the proteins in the cell using an intrabodywhich is a variable domain of kappa chain of human antibodies thatspecifically bind to intracellular proteins.

DETAILED DESCRIPTION OF THE INVENTION Technical Problems

However, those methods for analyzing protein interaction havedisadvantages in that the methods are not performed in real time andcannot eliminate the possibility of false positive or false-negative aswell as they are inefficient in that introducing specific mutations orscreening specific inhibitor for proteins are cost and time-consumingprocess. Moreover, the treatment of the specific inhibitor causesirreversible protein inactivation and thus it is difficult to analyzefunction of proteins essential for cell survival using the specificinhibitor.

The present invention intended to solve several problems, including theabove-mentioned problems, the purpose of the present invention is toprovide a method of forming reversible protein nano-cluster in cellsusing light.

In addition, the other purpose of the present invention is to provide amethod for analyzing inter-protein interaction and a kit using themethod for forming reversible protein nano-cluster.

Furthermore, another purpose of the present invention is to providemethods and kits for inhibiting a target protein in a cell or a subjectusing the formation of light-induced protein nano-cluster. However,these purposes are to be exemplary and the scope of the presentinvention is not limited thereto.

SUMMARY OF THE INVENTION

In an aspect to the present invention, an expression vector comprising apolynucleotide encoding a fusion protein including a light-inducedheterodimerized protein and a self-assembled protein is provided.

According to the expression vector, the light-induced heterodimerizedprotein may be CIB, CIBN, PhyB, PIF, FKF1, GIGANTEA, CRY, or PHR.

According to the expression vector, the self-assembled protein may beferritin, virus capsid protein, ferritin-like protein,calcium/calmodulin-dependent protein kinase II alpha subunit (CaMKIIα)or DsRed and the virus capsid protein may be a capsid protein derivedfrom CCMV (cowpea chlorotic mottle virus), Norwalk virus, SV40, or HPV(human papilloma virus). The description for the self-assembled proteinis applied to a first self-assembled protein and a second self-assembledprotein which will be described later.

According to the expression vector, the fusion protein may contain afluorescent protein, and the fluorescent protein may be added to theN-terminus or the C-termius of the fusion protein or the fluorescentprotein may be inserted between the light-induced dimerizing protein andthe self-assembled protein. The fluorescent protein may be greenfluorescent protein (GFP), yellow fluorescent protein (YFP), redfluorescent protein (RFP), orange fluorescent protein, cyan fluorescentprotein (CFP), blue fluorescent protein (BFP), or tetracysteinefluorescent motif. The green fluorescent protein may be EGFP (enhancedgreen fluorescent protein), Emerald (Tsien, Annu. Rev. Biochem., 67:509-544, 1998), Superfolder (Pedelacq et al., Nat. Biotech., 24: 79-88,2006), GFP (Prendergast et at, Biochem., 17 (17): 3448-3453, 1978),Azami Green (Karasawa, et al., J. Biol. Chem., 278: 34167-34171, 2003),TagGFP (Evrogen, Russia), TurboGFP (Shagin et al., Mol. Biol. Evol., 21(5): 841-850, 2004), ZsGreen (Matz et al., Nat. Biotechnol., 17:969-973, 1999) or T-Sapphire (Zapata-Hommer et at, BMC Biotechnol., 3:5,2003). The yellow fluorescent protein may be EYFP (enhanced yellowfluorescent protein, Tsien, Annu. Rev. Biochem., 67: 509-544, 1998),Topaz (Hat et al., Ann. NY Acad. Sci., 1: 627-633, 2002), Venus (Nagaiet al., Nat. Biotechnol., 20(1): 87-90, 2002), mCitrine (Griesbeck etal., J. Biol. Chem., 276: 29188-29194, 2001), Ypet (Nguyet andDaugherty, Nat. Biotechnol., 23(3): 355-360, 2005), TagYFP (Evrogen,Russia), PhiYFP (Shagin et al., Mol. Biol. Evol., 21(5): 841-850, 2004),ZsYellow1 (Matz et al., Nat. Biotechnol., 17: 969-973, 1999), or mBanana(Shaner et al., Nat. Biotechnol., 22: 1567-1572, 2004). The redfluorescent protein may be mRuby (Kredel et al., PLoS ONE, 4(2): e4391,2009), mApple (Shaner et al., Nat. Methods, 5(6): 545-551, 2008),mStrawberry (Shaner et al., Nat. Biotechnol., 22: 1567-1572, 2004) andAsRed2 (Shanner et at, Nat. Biotechnol., 22: 1567-1572, 2004) or mRFP(Campbell et al., Proc. Natl. Acad. Sci. USA, 99(12): 7877-7882, 2002).The orange fluorescent protein may be Kusabira Orange (Karawawa et al.,Biochem. J. 381(Pt 1): 307-312, 2004), Kusabira Orange2 (MBLInternational Corp., Japan), mOrange (Shaner et al., Nat. Biotechnol.,22: 1567-1572, 2004), mOrange2 (Shaner et al., Nat. Biotechnol., 22:1567-1572, 2004), dTomato (Shaner et al., Nat. Biotechnol., 22:1567-1572, 2004), dTomato-Tandem (Shaner et al., Nat. Biotechnol., 22:1567-1572, 2004), TagRFP (Merzlyak et al., Nat. Methods, 4(7): 555-557,2007), TagRFP-T (Shaner et al., Nat. Methods, 5(6): 545-551, 2008),DsRed (Baird et al., Proc. Natl. Acad. Sci. USA, 97: 11984-11989, 1999),DsRed2 (Clontech, USA), DsRed-Express (Clontech, USA), DsRed-Monomer(Clontech, USA), or mTangerine (Shaner et al., Nat Biotechnol, 22:1567-1572, 2004 above). The cyan fluorescent protein may be ECFP(enhanced cyan fluorescent protein, Cubitt et al., Trends Biochem. Sci.,20: 448-455, 1995), mECFP (Ai et al., Biochem. J., 400(3): 531-540,2006), mCerulean (Koushik et al., Biophys. J., 91(12): L99-L101, 2006),CyPet (Nguyet and Daugherty, Nat. Biotechnol., 23 (3): 355-360, 2005),AmCyan1 (Matz et al., Nat. Biotechnol., 17: 969-973, 1999), Midori-IshiCyan (Karawawa et al., Biochem. J., 381(Pt 1): 307-312, 2004), TagCFP(Evrogen, Russia) or mTFP1, (Ai et at, Biochem. J., 400 (3): 531-540,2006). The blue fluorescent protein may be EBFP (enhanced bluefluorescent protein, Clontech, USA), EBFP2 (Ai et al., Biochemistry, 46(20): 5904-5910, 2007), Azurite (Mena et al., Nat. Biotechnol., 24:1569-1571, 2006) or mTagBFP (Subach et al., Chem. Biol., 15(10):1116-1124, 2008). The far red fluorescent protein may be mPlum (Wang etal., Proc. Natl. Acad. Sci. USA, 101: 16745-16749, 2004), mCherry(Shanner et al., Nat. Biotechnol., 22: 1567-1572, 2004), dKeima-Tandem(Kogure et al., Methods, 45(3): 223-226, 2008), JRed (Shagin et al.,Mol. Biol. Evol., 21(5): 841-850, 2004), mRaspberry (Shanner et al.,Nat. Biotechnol., 22: 1567-1572, 2004), HcRed1 (Fradkov et al., Biochem.J., 368(Pt 1): 17-21, 2002), HcRed-Tandem (Fradkov et al., Nat.Biotechnol., 22(3): 289-296, 2004), AQ143 (Shkrob et al., Biochem. J.,392: 649-654, 2005). The tetracysteine fluorescent motif may be apolypeptide including an amino acid sequence of Cys-Cys-Xaa-Xaa-Cys-Cys(SEQ ID NO: 1), wherein the Xaa is any one amino acid except cysteine.The description for the fluorescent proteins applied to a firstfluorescent protein, a second fluorescent proteins, a third fluorescentprotein and a fourth fluorescent protein which will be described laterin a same manner.

In another aspect to the present invention, a kit for forminglight-induced protein nano-clusters comprising a first expression vectorincluding a polynucleotide encoding a first fusion protein comprising alight-induced heterodimerized protein and a first self-assembledprotein; and

a second expression vector including a polynucleotide encoding a partnerprotein capable of forming a heterodimer with the light-inducedheterodimerizing protein, or a second fusion protein containing thepartner protein and a second self-assembled protein is provided.

According to the kit, the light-induced heterodimerized protein may beCIB, CIBN, PhyB, PIF, FKF1, GIGANTEA, CRY, or PHR, and the partnerprotein may be CRY or PHR when the light-induced heterodimerized proteinis CIB or CIBN, the partner protein may be PIF when the light-inducedheterodimerized protein is PhyB, the partner protein may be GIFANTEAwhen the light-induced heterodimerized protein is FKF1, the partnerprotein may be CIB, CIBN, PhyB, PIF, FKF1, GIGANTEA, CRY, or PHR, andthe partner protein may be CRY or PHR when the light-inducedheterodimerized protein is CIB or CIBN, the partner protein may be PIFwhen the light-induced heterodimerized protein is PhyB, the partnerprotein may be GIFANTEA when the light-induced heterodimerized proteinis FKF1, the partner protein may be CIB or CIBN when the light-inducedheterodimerized protein is CRY or PHR, the partner protein may be PIFwhen the light-induced heterodimerized protein is PhyB, or the partnerprotein may be FKF1 when the light-induced heterodimerized protein isGIGANTEA. The PIF may be PIF3 or PIF6. Among the light-inducedheterodimerized protein or the partner protein, CRY or PHR may behomodimerized regardless of light irradiation.

According to the kit, the first self-assembled protein and the secondself-assembled protein may be independently ferritin, virus capsidprotein, ferritin-like protein, calcium/calmodulin-dependent proteinkinase II alpha subunit (CaMKIIα) or DsRed and the virus capsid proteinmay be a capsid protein derived from CCMV (cowpea chlorotic mottlevirus), Norwalk virus, SV40, or HPV (human papilloma virus). However,the first self-assembled protein and the second self-assembling proteinmay be same or different when they are used at the same time.

According to the kit, at least one among the first fusion protein, thepartner protein and the second fusion protein may further comprise afluorescent protein and the fluorescent protein may be added toN-termius or C-terminus of the first fusion protein, the partner proteinand/or the second fusion protein or may be inserted between thelight-induced heterodimerized protein and the first self-assembledprotein or between the partner protein and the second self-assembledprotein. At this time, the fluorescent protein may be green fluorescentprotein (GFP), yellow fluorescent protein (YFP), red fluorescent protein(RFP), orange fluorescent protein, cyan fluorescent protein (CFP), bluefluorescent protein (BFP), or tetracysteine fluorescent motif.

In another aspect to the present invention, a method for forming aprotein nano-cluster is provided, wherein the method comprises:providing a first expression vector including a polynucleotide encodinga first fusion protein comprising a light-induced heterodimerizedprotein and a first self-assembled protein, and a second expressionvector including a polynucleotide encoding a partner protein capable offorming a heterodimer with the light-induced heterodimerizing protein,or a second fusion protein containing the partner protein and a secondself-assembled protein;

transforming a cell, a tissue or a subject with the first expressionvector and the second expression vector;

irradiating light having wavelength capable of indcucingheterodimerization between the light-induced heterodimerized protein andthe partner protein to the cell, the tissue or the subject.

According to the method, the light-induced heterodimerized protein maybe CIB, CIBN, PhyB, PIF, FKF1, GIGANTEA, CRY, or PHR, and the partnerprotein may be CRY or PHR when the light-induced heterodimerized proteinis CIB or CIBN, the partner protein may be PIF when the light-inducedheterodimerized protein is PhyB, the partner protein may be GIFANTEAwhen the light-induced heterodimerized protein is FKF1, the partnerprotein may be CIB, CIBN, PhyB, PIF, FKF1, GIGANTEA, CRY, or PHR, andthe partner protein may be CRY or PHR when the light-inducedheterodimerized protein is CIB or CIBN, the partner protein may be PIFwhen the light-induced heterodimerized protein is PhyB, the partnerprotein may be GIFANTEA when the light-induced heterodimerized proteinis FKF1, the partner protein may be CIB or CIBN when the light-inducedheterodimerized protein is CRY or PHR, the partner protein may be PIFwhen the light-induced heterodimerized protein is PhyB, or the partnerprotein may be FKF1 when the light-induced heterodimerized protein isGIGANTEA. The PIF may be PIF3 or PIF6. Among the light-inducedheterodimerized protein or the partner protein, CRY or PHR may behomodimerized regardless of light irradiation.

According to the method, the first self-assembled protein and the secondself-assembled protein may be independently ferritin, virus capsidprotein, ferritin-like protein, calcium/calmodulin-dependent proteinkinase II alpha subunit (CaMKIIα) or DsRed and the virus capsid proteinmay be a capsid protein derived from CCMV (cowpea chlorotic mottlevirus), Norwalk virus, SV40, or HPV (human papilloma virus). However,the first self-assembled protein and the second self-assembling proteinmay be same or different when they are used at the same time.

According to the kit, at least one among the first fusion protein, thepartner protein and the second fusion protein may further comprises afluorescent protein and the fluorescent protein may be added toN-termius or C-terminus of the first fusion protein, the partner proteinand/or the second fusion protein or may be inserted between thelight-induced heterodimerized protein and the first self-assembledprotein or between the partner protein and the second self-assembledprotein. The fluorescent protein may be green fluorescent protein (GFP),yellow fluorescent protein (YFP), red fluorescent protein (RFP), orangefluorescent protein, cyan fluorescent protein (CFP), blue fluorescentprotein (BFP), or tetracysteine fluorescent motif.

In another aspect to the present invention, a method for analyzinginteraction between proteins using light-induced nano-cluster formationis provided, wherein the method comprises: expressing 1) a first fusionprotein comprising a first self-assembled protein, a first fluorescentprotein and a light-induced heterodimerized protein, 2) optionally asecond fusion protein comprising a second fluorescent protein and asecond self-assembled protein, 3) a third fusion protein comprising athird fluorescent protein, 4) a partner protein capable ofheterodimerizing with light-induced heterodimerized protein by lightirradiating, 5) a bait protein, and 6) a target protein being analyzedwhether it interacts with the bait protein or not in a cell at the sametime, wherein the partner protein is incorporated to the second fusionprotein or the third fusion protein, the bait protein is incorporated toone of the second fusion protein, the third protein and the fourthfusion protein containing the first self-assembled protein and the firstfluorescent protein, and the target protein is incorporated to one ofother fusion proteins except the fusion protein containing the baitprotein or a fifth fusion protein comprising a fourth fluorescentprotein;

irradiating light having wavelength capable of indcucingheterodimerization between the light-induced heterodimerized protein andthe partner protein to the cell; and

observing the pattern and intensity of fluorescence of the fluorescentproteins,

with the proviso the second fluorescent protein may be omitted in casethat light-induced heterodimerized protein or the partner protein formsa homodimer regardless of light irradiating, wherein all the fluorescentproteins emit different wavelengths of light, and wherein the firstself-assembled protein or the second self-assembled protein may beomitted form any fusion proteins containing DsRed when either the firstfluorescent protein or the second fluorescent protein is DsRed.

According to the method, the first fluorescent to the fourth fluorescentprotein may be independently green fluorescent protein (GFP), yellowfluorescent protein (YFP), red fluorescent protein (RFP), orangefluorescent protein, cyan fluorescent protein (CFP), blue fluorescentprotein (BFP), or tetracysteine fluorescent motif.

According to the method, the first self-assembled protein and the secondself-assembled protein may be independently ferritin, virus capsidprotein, ferritin-like protein, calcium/calmodulin-dependent proteinkinase II alpha subunit (CaMKIIα) or DsRed and the virus capsid proteinmay be a capsid protein derived from CCMV (cowpea chlorotic mottlevirus), Norwalk virus, SV40, or HPV (human papilloma virus). However,the first self-assembled protein and the second self-assembling proteinmay be same or different when they are used at the same time.

According to the method, the light-induced heterodimerized protein maybe CIB, CIBN, PhyB, PIF, FKF1, GIGANTEA, CRY, or PHR, and the partnerprotein may be CRY or PHR when the light-induced heterodimerized proteinis CIB or CIBN, the partner protein may be PIF when the light-inducedheterodimerized protein is PhyB, the partner protein may be GIFANTEAwhen the light-induced heterodimerized protein is FKF1, the partnerprotein may be CIB, CIBN, PhyB, PIF, FKF1, GIGANTEA, CRY, or PHR, andthe partner protein may be CRY or PHR when the light-inducedheterodimerized protein is CIB or CIBN, the partner protein may be PIFwhen the light-induced heterodimerized protein is PhyB, the partnerprotein may be GIFANTEA when the light-induced heterodimerized proteinis FKF1, the partner protein may be CIB or CIBN when the light-inducedheterodimerized protein is CRY or PHR, the partner protein may be PIFwhen the light-induced heterodimerized protein is PhyB, or the partnerprotein may be FKF1 when the light-induced heterodimerized protein isGIGANTEA. The PIF may be PIF3 or PIF6. Among the light-inducedheterodimerized protein or the partner protein, CRY or PHR may behomodimerized regardless of light irradiation.

In another aspect to the present invention, a method for analyzinginteraction between proteins using light-induced nano-cluster formationis provided, wherein the method comprises: expressing 1) a first fusionprotein comprising a first self-assembled protein, a first fluorescentprotein and a light-induced heterodimerized protein, 2) optionally asecond fusion protein comprising a second fluorescent protein and asecond self-assembled protein, 3) a third fusion protein comprising athird fluorescent protein, 4) a partner protein capable ofheterodimerizing with light-induced heterodimerized protein by lightirradiating, 5) a bait protein, and 6) a target protein interacting withthe bait protein in a cell at the same time, wherein the partner proteinis incorporated to the second fusion protein or the third fusionprotein, the bait protein is incorporated to one of the second fusionprotein, the third protein and the fourth fusion protein containing thefirst self-assembled protein and the first fluorescent protein, and thetarget protein is incorporated to one of other fusion proteins exceptthe fusion protein containing the bait protein or a fifth fusion proteincomprising a fourth fluorescent protein;

treating a candidate substance capable of controlling the interactionbetween the bait protein and the target protein to the cell;

irradiating light having wavelength capable of indcucingheterodimerization between the light-induced heterodimerized protein andthe partner protein to the cell before, after or at the same time oftreating the candidate substance to the cell; and

observing the pattern and intensity of fluorescence of the fluorescentproteins,

with the proviso that the second fluorescent protein may be omitted incase that light-induced heterodimerized protein or the partner proteinforms a homodimer regardless of light irradiating, wherein all thefluorescent proteins emit different wavelengths of light, and whereinthe first self-assembled protein or the second self-assembled proteinmay be omitted from any fusion proteins containing DsRed when either thefirst fluorescent protein or the second fluorescent protein is DsRed.

According to the method, the first fluorescent to the fourth fluorescentprotein may be independently green fluorescent protein (GFP), yellowfluorescent protein (YFP), red fluorescent protein (RFP), orangefluorescent protein, cyan fluorescent protein (CFP), blue fluorescentprotein (BFP), or tetracysteine fluorescent motif.

According to the method, the first self-assembled protein and the secondself-assembled protein may be independently ferritin, virus capsidprotein, ferritin-like protein, calcium/calmodulin-dependent proteinkinase II alpha subunit (CaMKIIα) or DsRed and the virus capsid proteinmay be a capsid protein derived from CCMV (cowpea chlorotic mottlevirus), Norwalk virus, SV40, or HPV (human papilloma virus). However,the first self-assembled protein and the second self-assembling proteinmay be same or different when they are used at the same time.

According to the method, the light-induced heterodimerized protein maybe CIB, CIBN, PhyB, PIF, FKF1, GIGANTEA, CRY, or PHR, and the partnerprotein may be CRY or PHR when the light-induced heterodimerized proteinis CIB or CIBN, the partner protein may be PIF when the light-inducedheterodimerized protein is PhyB, the partner protein may be GIFANTEAwhen the light-induced heterodimerized protein is FKF1, the partnerprotein may be CIB, CIBN, PhyB, PIF, FKF1, GIGANTEA, CRY, or PHR, andthe partner protein may be CRY or PHR when the light-inducedheterodimerized protein is CIB or CIBN, the partner protein may be PIFwhen the light-induced heterodimerized protein is PhyB, the partnerprotein may be GIFANTEA when the light-induced heterodimerized proteinis FKF1, the partner protein may be CIB or CIBN when the light-inducedheterodimerized protein is CRY or PHR, the partner protein may be PIFwhen the light-induced heterodimerized protein is PhyB, or the partnerprotein may be FKF1 when the light-induced heterodimerized protein isGIGANTEA. The PIF may be PIF3 or PIF6. Among the light-inducedheterodimerized protein or the partner protein, CRY or PHR may behomodimerized regardless of light irradiation.

In another aspect of the present invention, a kit for analyzinginteraction between proteins is provided, wherein the kit comprises:

a first expression vector including a first gene construct containing apromoter and a polynucleotide encoding a first fusion protein comprisinga first self-assembled protein, a first fluorescent protein and alight-induced heterodimerized protein, wherein the polynucleotide isoperably linked to the promoter;

optionally a second expression vector including a second gene constructcontaining a promoter and a polynucleotide encoding a second fusionprotein comprising a second fluorescent protein and a secondself-assembled protein, wherein the polynucleotide is operably linked tothe promoter;

a third expression vector including a third gene construct containing apromoter and a polynucleotide encoding a third fusion protein comprisinga third fluorescent protein, wherein the polynucleotide is operablylinked to the promoter; and

optionally a fourth expression vector including a fourth gene constructcontaining a promoter and a polynucleotide encoding a fourth fusionprotein containing the first self-assembled protein, the firstfluorescent protein and a bait protein, wherein the polynucleotide isoperably linked to the promoter,

wherein the bait protein is incorporated to one of the second fusionprotein and the third fusion protein when the fourth expression vectoris omitted, and one of the second fusion protein and the third fusionprotein contains a partner protein capable of heterodimerizing withlight-induced heterodimerized protein by light irradiating, one lackingthe bait protein among the second fusion protein and the third fusionprotein or a fifth expression vector optionally expressed whichcomprises a fifth fusion protein comprising a fourth fluorescent proteincontains a target protein which is a subject to be examined whether itinteracts with the bait protein or not,

with the proviso that the second fluorescent protein may be omitted incase that light-induced heterodimerized protein or the partner proteinforms a homodimer regardless of light irradiating, wherein all thefluorescent proteins emit different wavelengths of light, and whereinthe first self-assembled protein or the second self-assembled proteinmay be omitted from any fusion proteins containing DsRed when either thefirst fluorescent protein or the second fluorescent protein is DsRed.

According to the kit, the first fluorescent to the fourth fluorescentprotein may be independently green fluorescent protein (GFP), yellowfluorescent protein (YFP), red fluorescent protein (RFP), orangefluorescent protein, cyan fluorescent protein (CFP), blue fluorescentprotein (BFP), or tetracysteine fluorescent motif.

According to the method, the first self-assembled protein and the secondself-assembled protein may be independently ferritin, virus capsidprotein, ferritin-like protein, calcium/calmodulin-dependent proteinkinase II alpha subunit (CaMKIIα) or DsRed and the virus capsid proteinmay be a capsid protein derived from CCMV (cowpea chlorotic mottlevirus), Norwalk virus, SV40, or HPV (human papilloma virus). However,the first self-assembled protein and the second self-assembling proteinmay be same or different when they are used at the same time.

According to the method, the light-induced heterodimerized protein maybe CIB, CIBN, PhyB, PIF, FKF1, GIGANTEA, CRY, or PHR, and the partnerprotein may be CRY or PHR when the light-induced heterodimerized proteinis CIB or CIBN, the partner protein may be PIF when the light-inducedheterodimerized protein is PhyB, the partner protein may be GIFANTEAwhen the light-induced heterodimerized protein is FKF1, the partnerprotein may be CIB, CIBN, PhyB, PIF, FKF1, GIGANTEA, CRY, or PHR, andthe partner protein may be CRY or PHR when the light-inducedheterodimerized protein is CIB or CIBN, the partner protein may be PIFwhen the light-induced heterodimerized protein is PhyB, the partnerprotein may be GIFANTEA when the light-induced heterodimerized proteinis FKF1, the partner protein may be CIB or CIBN when the light-inducedheterodimerized protein is CRY or PHR, the partner protein may be PIFwhen the light-induced heterodimerized protein is PhyB, or the partnerprotein may be FKF1 when the light-induced heterodimerized protein isGIGANTEA. The PIF may be PIF3 or PIF6. Among the light-inducedheterodimerized protein or the partner protein, CRY or PHR may behomodimerized regardless of light irradiation.

In another aspect of the present invention, a kit for analyzinginteraction between proteins is provided, wherein the kit comprises:

a first expression vector including a first gene construct containing apromoter and a first polynucleotide encoding a first fusion proteincomprising a first self-assembled protein, a first fluorescent proteinand a light-induced heterodimerized protein, wherein the polynucleotideis operably linked to the promoter;

optionally a second expression vector including a second gene constructcontaining a promoter and a second polynucleotide encoding a secondfusion protein comprising a second fluorescent protein and a secondself-assembled protein, wherein the second polynucleotide is operablylinked to the promoter;

a third expression vector including a third gene construct containing apromoter and a third polynucleotide encoding a third fusion proteincomprising a third fluorescent protein, wherein the third polynucleotideis operably linked to the promoter; and

optionally a fourth expression vector including a fourth gene constructcomprising a fourth polynucleotide encoding a fourth fusion proteincontaining the first self-assembled protein and the first fluorescentprotein and a multicloning site for inserting a polynucleotide encodinga bait protein to the fourth polynucleotide, wherein the polynucleotideencoding a bait protein is linked operably to the fourth polyncleotide,

wherein the multicloning site is incorporated to one of the secondexpression vector and the third expression when the fourth expressionvector is omitted, and one of the second fusion protein and the thirdfusion protein contains a partner protein capable of heterodimerizingwith light-induced heterodimerized protein by light irradiating, anwherein one lacking the multicloning site for inserting a polynucleotideencoding a bait protein among the second expression vector and the thirdexpression vector or an optionally expressed fifth expression vectorincluding a fifth polynucleotide encoding a fifth fusion proteincomprising a fourth fluorescent protein contains a multicloning site forinserting a polynucleotide encoding a target protein which is a subjectto be examined whether it interacts with the bait protein or not,

with the proviso that the second fluorescent protein may be omitted incase that light-induced heterodimerized protein or the partner proteinforms a homodimer regardless of light irradiating, wherein all thefluorescent proteins emit different wavelengths of light, and whereinthe first self-assembled protein or the second self-assembled proteinmay be omitted when either the first fluorescent protein or the secondfluorescent protein is DsRed.

According to the kit, the first fluorescent to the fourth fluorescentprotein may be independently green fluorescent protein (GFP), yellowfluorescent protein (YFP), red fluorescent protein (RFP), orangefluorescent protein, cyan fluorescent protein (CFP), blue fluorescentprotein (BFP), or tetracysteine fluorescent motif.

According to the method, the first self-assembled protein and the secondself-assembled protein may be independently ferritin, virus capsidprotein, ferritin-like protein, calcium/calmodulin-dependent proteinkinase II alpha subunit (CaMKIIα) or DsRed and the virus capsid proteinmay be a capsid protein derived from CCMV (cowpea chlorotic mottlevirus), Norwalk virus, SV40, or HPV (human papilloma virus). However,the first self-assembled protein and the second self-assembling proteinmay be same or different when they are used at the same time.

According to the method, the light-induced heterodimerized protein maybe CIB, CIBN, PhyB, PIF, FKF1, GIGANTEA, CRY, or PHR, and the partnerprotein may be CRY or PHR when the light-induced heterodimerized proteinis CIB, CIBN, PhyB, PIF, FKF1, GIGANTEA, CRY, or PHR, and the partnerprotein may be CRY or PHR when the light-induced heterodimerized proteinis CIB or CIBN, the partner protein may be PIF when the light-inducedheterodimerized protein is PhyB, the partner protein may be GIFANTEAwhen the light-induced heterodimerized protein is FKF1, the partnerprotein may be CIB or CIBN when the light-induced heterodimerizedprotein is CRY or PHR, the partner protein may be PIF when thelight-induced heterodimerized protein is PhyB, or the partner proteinmay be FKF1 when the light-induced heterodimerized protein is GIGANTEA.The PIF may be PIF3 or PIF6. Among the light-induced heterodimerizedprotein or the partner protein, CRY or PHR may be homodimerizedregardless of light irradiation.

In another aspect of the present invention, a method for inhibiting atarget protein reversibly using light-induced protein nano-clusterformation is provided, wherein the method comprises: expressing a fusionprotein comprising a self-assembled protein and a light-inducedheterodimerized protein, a partner protein capable of heterodimerizingwith light-induced heterodimerized protein and a bait proteininteracting with a target protein in a cell or a subject expressing thetarget protein as an inherent protein; and Inducing a proteinnano-cluster formation by irradiating light having wavelength capable ofinducing heterodimerizing between the light-induced heterodimerizedprotein and the partner protein to the cell or the subject.

According to the method, the self-assembled protein may be independentlyferritin, virus capsid protein, ferritin-like protein,calcium/calmodulin-dependent protein kinase II alpha subunit (CaMKIIα)or DsRed and the virus capsid protein may be a capsid protein derivedfrom CCMV (cowpea chlorotic mottle virus), Norwalk virus, SV40, or HPV(human papilloma virus).

According to the method, at least one protein among the fusion protein,the partner protein and the bait protein may contain a fluorescentprotein in order to verify whether the protein nano-cluster is formed.At this time, the fluorescent protein may be green fluorescent protein(GFP), yellow fluorescent protein (YFP), red fluorescent protein (RFP),orange fluorescent protein, cyan fluorescent protein (CFP), bluefluorescent protein (BFP), far-red fluorescent protein or tetracysteinefluorescent motif.

According to the method, the light-induced heterodimerized protein maybe CIB, CIBN, PhyB, PIF, FKF1, GIGANTEA, CRY, or PHR, and the partnerprotein may be CRY or PHR when the light-induced heterodimerized proteinis CIB or CIBN, the partner protein may be PIF when the light-inducedheterodimerized protein is PhyB, the partner protein may be GIFANTEAwhen the light-induced heterodimerized protein is FKF1, the partnerprotein may be CIB or CIBN when the light-induced heterodimerizedprotein is CRY or PHR, the partner protein may be PIF when thelight-induced heterodimerized protein is PhyB, or the partner proteinmay be FKF1 when the light-induced heterodimerized protein is GIGANTEA.The PIF may be PIF3 or PIF6.

According to the method, the light-induced heterodimerized protein orthe partner protein may be homodimerized regardless of lightirradiation. In this case the light-induced heterodimerized protein orthe partner protein capable of forming homodimer regardless of lightirradiation may be CRY or PHR.

In another aspect of the present invention, a method for inhibiting atarget protein reversibly using light-induced protein nano-clusterformation is provided, wherein the method comprises:co-expressing afirst fusion protein comprising a first self-assembled protein and alight-induced heterodimerized protein, a second fusion proteincomprising a second self-assembled protein and a partner protein capableof heterodimerizing with light-induced heterodimerized protein and abait protein interacting with a target protein in a cell or a subjectexpressing the target protein as an inherent protein; and inducing aprotein nano-cluster formation by irradiating light having wavelengthcapable of inducing heterodimerizing between the light-inducedheterodimerized protein and the partner protein to the cell or thesubject, wherein the bait protein is expressed as a fusion protein withthe first self-assembled protein or the second self-assembled protein.

According to the method, the first self-assembled protein and the secondself-assembled protein may be independently ferritin, virus capsidprotein, ferritin-like protein, calcium/calmodulin-dependent proteinkinase II alpha subunit (CaMKIIα) or DsRed and the virus capsid proteinmay be a capsid protein derived from CCMV (cowpea chlorotic mottlevirus), Norwalk virus, SV40, or HPV (human papilloma virus).

According to the method, at least one protein among the first fusionprotein, the second fusion protein, the partner protein and the baitprotein may contain a fluorescent protein in order to verify whether theprotein nano-cluster is formed. At this time, the fluorescent proteinmay be green fluorescent protein (GFP), yellow fluorescent protein(YFP), red fluorescent protein (RFP), orange fluorescent protein, cyanfluorescent protein (CFP), blue fluorescent protein (BFP), far-redfluorescent protein or tetracysteine fluorescent motif.

According to the method, the light-induced heterodimerized protein maybe CIB, CIBN, PhyB, PIF, FKF1, GIGANTEA, CRY, or PHR, and the partnerprotein may be CRY or PHR when the light-induced heterodimerized proteinis CIB or CIBN, the partner protein may be PIF when the light-inducedheterodimerized protein is PhyB, the partner protein may be GIFANTEAwhen the light-induced heterodimerized protein is FKF1, the partnerprotein may be CIB or CIBN when the light-induced heterodimerizedprotein is CRY or PHR, the partner protein may be PIF when thelight-induced heterodimerized protein is PhyB, or the partner proteinmay be FKF1 when the light-induced heterodimerized protein is GIGANTEA.The PIF may be PIF3 or PIF6.

According to the method, the light-induced heterodimerized protein orthe partner protein may be homodimerized regardless of lightirradiation. In this case the light-induced heterodimerized protein orthe partner protein capable of forming homodimer regardless of lightirradiation may be CRY or PHR.

In another aspect of the present invention, a kit for inhibiting atarget protein reversibly using light-induced protein nano-clusterformation is provided, wherein the kit comprises: a first expressionvector including a first gene construct containing a promoter and afirst polynucleotide encoding a fusion protein comprising aself-assembled protein and a light-induced heterodimerized protein,wherein the first polynucleotide is operably linked to the promoter; asecond expression vector including a second gene construct containing apromoter and a second polynucleotide encoding a partner protein capableof heterodimerizing with light-induced heterodimerized protein, whereinthe second polynucleotide is operably linked to the promoter, and athird expression vector including a third gene construct containing apromoter and a third polynucleotide encoding a bait protein interactingwith the target protein, wherein the third polynucleotide is operablylinked to the promoter, wherein the bait protein is expressed as afusion protein with the self-assembled protein or the partner protein.

According to the kit, the first self-assembled protein and the secondself-assembled protein may be independently ferritin, virus capsidprotein, ferritin-like protein, calcium/calmodulin-dependent proteinkinase II alpha subunit (CaMKIIα) or DsRed and the virus capsid proteinmay be a capsid protein derived from CCMV (cowpea chlorotic mottlevirus), Norwalk virus, SV40, or HPV (human papilloma virus).

According to the kit, at least one protein among the first fusionprotein, the second fusion protein, the partner protein and the baitprotein may contain a fluorescent protein in order to verify whether theprotein nano-cluster is formed. At this time, the fluorescent proteinmay be green fluorescent protein (GFP), yellow fluorescent protein(YFP), red fluorescent protein (RFP), orange fluorescent protein, cyanfluorescent protein (CFP), blue fluorescent protein (BFP), far-redfluorescent protein or tetracysteine fluorescent motif.

According to the kit, the light-induced heterodimerized protein may beCIB, CIBN, PhyB, PIF, FKF1, GIGANTEA, CRY, or PHR, and the partnerprotein may be CRY or PHR when the light-induced heterodimerized proteinis CIB or CIBN, the partner protein may be PIF when the light-inducedheterodimerized protein is PhyB, the partner protein may be GIFANTEAwhen the light-induced heterodimerized protein is FKF1, the partnerprotein may be CIB or CIBN when the light-induced heterodimerizedprotein is CRY or PHR, the partner protein may be PIF when thelight-induced heterodimerized protein is PhyB, or the partner proteinmay be FKF1 when the light-induced heterodimerized protein is GIGANTEA.The PIF may be PIF3 or PIF6.

According to the method, the light-induced heterodimerized protein orthe partner protein may be homodimerized regardless of lightirradiation. In this case the light-induced heterodimerized protein orthe partner protein capable of forming homodimer regardless of lightirradiation may be CRY or PHR.

In another aspect of the present invention, a kit for inhibiting atarget protein reversibly using light-induced protein nano-clusterformation is provided, wherein the kit comprises: a first expressionvector including a first gene construct containing a promoter and afirst polynucleotide encoding a first fusion protein comprising a firstself-assembled protein and a light-induced heterodimerized protein,wherein the first polynucleotide is operably linked to the promoter; asecond expression vector including a second gene construct containing apromoter and a second polynucleotide encoding a second fusion proteincomprising a second self-assembled protein and a partner protein capableof heterodimerizing with the light-induced heterodimerized protein,wherein the second polynucleotide is operably linked to the promoter;and a third expression vector including a third gene constructcontaining a promoter and a multicloning site for inserting a thirdpolynucleotide encoding a bait protein interacting with the targetprotein, wherein the third polynucleotide is operably linked to thepromoter, wherein the bait protein is expressed as a fusion protein withthe self-assembled protein or the partner protein.

According to the kit, the first self-assembled protein and the secondself-assembled protein may be independently ferritin, virus capsidprotein, ferritin-like protein, calcium/calmodulin-dependent proteinkinase II alpha subunit (CaMKIIα) or DsRed and the virus capsid proteinmay be a capsid protein derived from CCMV (cowpea chlorotic mottlevirus), Norwalk virus, SV40, or HPV (human papilloma virus).

According to the kit, at least one protein among the first fusionprotein, the second fusion protein, the partner protein and the baitprotein may contain a fluorescent protein in order to verify whether theprotein nano-cluster is formed. At this time, the fluorescent proteinmay be green fluorescent protein (GFP), yellow fluorescent protein(YFP), red fluorescent protein (RFP), orange fluorescent protein, cyanfluorescent protein (CFP), blue fluorescent protein (BFP), far-redfluorescent protein or tetracysteine fluorescent motif.

According to the kit, the light-induced heterodimerized protein may beCIB, CIBN, PhyB, PIF, FKF1, GIGANTEA, CRY, or PHR, and the partnerprotein may be CRY or PHR when the light-induced heterodimerized proteinis CIB or CIBN, the partner protein may be PIF when the light-inducedheterodimerized protein is PhyB, the partner protein may be GIFANTEAwhen the light-induced heterodimerized protein is FKF1, the partnerprotein may be CIB or CIBN when the light-induced heterodimerizedprotein is CRY or PHR, the partner protein may be PIF when thelight-induced heterodimerized protein is PhyB, or the partner proteinmay be FKF1 when the light-induced heterodimerized protein is GIGANTEA.The PIF may be PIF3 or PIF6.

According to the kit, the light-induced heterodimerized protein or thepartner protein may be homodimerized regardless of light irradiation. Inthis case the light-induced heterdimerizable protein or the partnerprotein capable of forming homodimer regardless of light irradiation maybe CRY or PHR.

In another aspect of the present invention, a kit for inhibiting atarget protein reversibly using light-induced protein nano-clusterformation is provided, wherein the kit comprises: a first expressionvector including a first gene construct containing a promoter and afirst polynucleotide encoding a fusion protein comprising aself-assembled protein and a light-induced heterodimerized protein,wherein the first polynucleotide is operably linked to the promoter, asecond expression vector including a second gene construct containing apromoter and a second polynucleotide encoding a partner protein capableof heterodimerizing with the light-induced heterodimerized protein,wherein the second polynucleotide is operably linked to the promoter;and a third expression vector including a third gene constructcontaining a promoter and a multicloning site for inserting a thirdpolynucleotide encoding a bait protein interacting with the targetprotein, wherein the third polynucleotide is operably linked to thepromoter, wherein the bait protein is expressed as a fusion protein withthe self-assembled protein or the partner protein.

According to the kit, the self-assembled protein may be ferritin, viruscapsid protein, ferritin-like protein, calcium/calmodulin-dependentprotein kinase II alpha subunit (CaMKIIα) or DsRed and the virus capsidprotein may be a capsid protein derived from CCMV (cowpea chloroticmottle virus), Norwalk virus, SV40, or HPV (human papilloma virus).

According to the kit, at least one protein among the fusion protein, thepartner protein and the bait protein may contain a fluorescent proteinin order to verify whether the protein nano-cluster is formed. At thistime, the fluorescent protein may be green fluorescent protein (GFP),yellow fluorescent protein (YFP), red fluorescent protein (RFP), orangefluorescent protein, cyan fluorescent protein (CFP), blue fluorescentprotein (BFP), far-red fluorescent protein or tetracysteine fluorescentmotif.

According to the kit, the light-induced heterodimerized protein may beCIB, CIBN, PhyB, PIF, FKF1, GIGANTEA, CRY, or PHR, and the partnerprotein may be CRY or PHR when the light-induced heterodimerized proteinis CIB or CIBN, the partner protein may be PIF when the light-inducedheterodimerized protein is PhyB, the partner protein may be GIFANTEAwhen the light-induced heterodimerized protein is FKF1, the partnerprotein may be CIB or CIBN when the light-induced heterodimerizedprotein is CRY or PHR, the partner protein may be PIF when thelight-induced heterodimerized protein is PhyB, or the partner proteinmay be FKF1 when the light-induced heterodimerized protein is GIGANTEA.The PIF may be PIF3 or PIF6.

According to kit, the light-induced heterodimerized protein or thepartner protein may be homodimerized regardless of light irradiation. Inthis case, the light-induced heterodimerized protein or the partnerprotein capable of forming homodimer regardless of light irradiation maybe CRY or PHR.

In another aspect of the present invention, a kit for inhibiting atarget protein reversibly using light-induced protein nano-clusterformation is provided, wherein the kit comprises: a first expressionvector including a first gene construct containing a promoter and afirst polynucleotide encoding a first fusion protein comprising a firstself-assembled protein and a light-induced heterodimerized protein,wherein the first polynucleotide is operably linked to the promoter; asecond expression vector including a second gene construct containing apromoter and a second polynucleotide encoding a second fusion proteincomprising a second self-assembled protein and a partner protein capableof heterodimerizing with the light-induced heterodimerized protein,wherein the second polynucleotide is operably linked to the promoter;and a third expression vector including a third gene constructcomprising a promoter and a third polynucleotide encoding a bait proteininteracting with the target protein, wherein the third polynucleotide isoperably linked to the promoter, wherein the bait protein is expressedas a fusion protein with the first self-assembled protein or the secondself-assembled protein.

According to the kit, the first self-assembled protein of the secondself-assembled protein may be ferritin, virus capsid protein,ferritin-like protein, calcium/calmodulin-dependent protein kinase IIalpha subunit (CaMKIIα) or DsRed and the virus capsid protein may be acapsid protein derived from CCMV (cowpea chlorotic mottle virus),Norwalk virus, SV40, or HPV (human papilloma virus).

According to the kit, at least one protein among the first fusionprotein, the second fusion protein, the partner protein and the baitprotein may contain a fluorescent protein in order to verify whether theprotein nano-cluster is formed. At this time, the fluorescent proteinmay be green fluorescent protein (GFP), yellow fluorescent protein(YFP), red fluorescent protein (RFP), orange fluorescent protein, cyanfluorescent protein (CFP), blue fluorescent protein (BFP), far-redfluorescent protein or tetracysteine fluorescent motif.

According to the kit, the light-induced heterodimerized protein may beCIB, CIBN, PhyB, PIF, FKF1, GIGANTEA, CRY, or PHR, and the partnerprotein may be CRY or PHR when the light-induced heterodimerized proteinis CIB or CIBN, the partner protein may be PIF when the light-inducedheterodimerized protein is PhyB, the partner protein may be GIFANTEAwhen the light-induced heterodimerized protein is FKF1, the partnerprotein may be CIB or CIBN when the light-induced heterodimerizedprotein is CRY or PHR, the partner protein may be PIF when thelight-induced heterodimerized protein is PhyB, or the partner proteinmay be FKF1 when the light-induced heterodimerized protein is GIGANTEA.The PIF may be PIF3 or PIF6.

According to kit, the light-induced heterodimerized protein or thepartner protein may be homodimerized regardless of light irradiation. Inthis case, the light-induced heterodimerized protein or the partnerprotein capable of forming homodimer regardless of light irradiation maybe CRY or PHR.

In an aspect of the present invention, a method for inhibiting a targetprotein reversibly using light-induced nanocluster formation, whereinthe method comprises: expressing a fusion protein includingself-assembled protein and a light-induced heterodimerzable protein, apartner protein forming a heterodimer with the light-inducedheterodimerzable protein, and a target protein in a cell or a subject;and inducing light-induced formation of nanocluster by light havingwavelength capable of forming the heterodimer between the light-inducedheterodimerized protein and the partner protein, wherein the targetprotein is expressed as a fusion protein with the self-assembled proteinor the partner protein.

According to the method, the self-assembled protein may be ferritin,virus capsid protein, ferritin-like protein,calcium/calmodulin-dependent protein kinase II alpha subunit (CaMKIIα)or DsRed and the virus capsid protein may be a capsid protein derivedfrom CCMV (cowpea chlorotic mottle virus), Norwalk virus, SV40, or HPV(human papilloma virus).

According to the method, at least one protein among the fusion protein,and the partner protein may contain a fluorescent protein in order toverify whether the protein nano-cluster is formed. At this time, thefluorescent protein may be green fluorescent protein (GFP), yellowfluorescent protein (YFP), red fluorescent protein (RFP), orangefluorescent protein, cyan fluorescent protein (CFP), blue fluorescentprotein (BFP), far-red fluorescent protein or tetracysteine fluorescentmotif.

According to the method, the light-induced heterodimerized protein maybe CIB, CIBN, PhyB, PIF, FKF1, GIGANTEA, CRY, or PHR, and the partnerprotein may be CRY or PHR when the light-induced heterodimerized proteinis CIB or CIBN, the partner protein may be PIF when the light-inducedheterodimerized protein is PhyB, the partner protein may be GIFANTEAwhen the light-induced heterodimerized protein is FKF1, the partnerprotein may be CIB or CIBN when the light-induced heterodimerizedprotein is CRY or PHR, the partner protein may be PIF when thelight-induced heterodimerized protein is PhyB, or the partner proteinmay be FKF1 when the light-induced heterodimerized protein is GIGANTEA.The PIF may be PIF3 or PIF6.

According to the method, the light-induced heterodimerized protein orthe partner protein may be homodimerized regardless of lightirradiation. In this case, the light-induced heterodimerized protein orthe partner protein capable of forming homodimer regardless of lightirradiation may be CRY or PHR.

In another aspect of the present invention, a kit for inhibiting atarget protein reversibly using light-induced protein nano-clusterformation is provided, wherein the kit comprises: a first expressionvector including a first gene construct containing a promoter and afirst polynucleotide encoding a fusion protein comprising aself-assembled protein and a light-induced heterodimerized protein,wherein the first polynucleotide is operably linked to the promoter; asecond expression vector including a second gene construct containing apromoter and a second polynucleotide encoding a second fusion proteincomprising a partner protein capable of heterodimerizing with thelight-induced heterodimerized protein, wherein the second polynucleotideis operably linked to the promoter; and optionally a third expressionvector including a third gene construct containing a promoter and athird polynucleotide encoding a third fusion protein containing thetarget protein and the self-assembled protein, wherein the thirdpolynucleotide is linked operably to the promoter, or the target proteinis included in the second fusion protein.

According to the kit, the self-assembled protein may be ferritin, viruscapsid protein, ferritin-like protein, calcium/calmodulin-dependentprotein kinase II alpha subunit (CaMKIIα) or DsRed and the virus capsidprotein may be a capsid protein derived from CCMV (cowpea chloroticmottle virus), Norwalk virus, SV40, or HPV (human papilloma virus).

According to the kit, at least one protein among the fusion protein, andthe partner protein may contain a fluorescent protein in order to verifywhether the protein nano-cluster is formed. At this time, thefluorescent protein may be green fluorescent protein (GFP), yellowfluorescent protein (YFP), red fluorescent protein (RFP), orangefluorescent protein, cyan fluorescent protein (CFP), blue fluorescentprotein (BFP), far-red fluorescent protein or tetracysteine fluorescentmotif.

According to the kit, the light-induced heterodimerized protein may beCIB, CIBN, PhyB, PIF, FKF1, GIGANTEA, CRY, or PHR, and the partnerprotein may be CRY or PHR when the light-induced heterodimerized proteinis CIB or CIBN, the partner protein may be PIF when the light-inducedheterodimerized protein is PhyB, the partner protein may be GIFANTEAwhen the light-induced heterodimerized protein is FKF1, the partnerprotein may be CIB or CIBN when the light-induced heterodimerizedprotein is CRY or PHR, the partner protein may be PIF when thelight-induced heterodimerized protein is PhyB, or the partner proteinmay be FKF1 when the light-induced heterodimerized protein is GIGANTEA.The PIF may be PIF3 or PIF6.

According to the kit, the light-induced heterodimerized protein or thepartner protein may be homodimerized regardless of light irradiation. Inthis case, the light-induced heterodimerized protein or the partnerprotein capable of forming homodimer regardless of light irradiation maybe CRY or PHR.

In another aspect of the present invention, a kit for inhibiting atarget protein reversibly using light-induced protein nano-clusterformation is provided, wherein the kit comprises: a first expressionvector including a first gene construct containing a promoter and afirst polynucleotide encoding a fusion protein comprising aself-assembled protein and a light-induced heterodimerized protein,wherein the first polynucleotide is operably linked to the promoter; asecond expression vector including a second gene construct containing apromoter and a second polynucleotide encoding a second fusion proteincomprising a partner protein capable of heterodimerizing with thelight-induced heterodimerized protein, wherein the second polynucleotideis operably linked to a promoter; and optionally a third expressionvector including a third gene construct containing a promoter and amulticloning site for inserting a third polynucleotide encoding a baitprotein interacting with the target protein, wherein the thirdpolynucleotide is operably linked to the promoter, or the multicloningsite is included within the second polynucleotide in order that thetarget protein is expressed as included in the second fusion protein.

According to the kit, the self-assembled protein may be ferritin, viruscapsid protein, ferritin-like protein, calcium/calmodulin-dependentprotein kinase II alpha subunit (CaMKIIα) or DsRed and the virus capsidprotein may be a capsid protein derived from CCMV (cowpea chloroticmottle virus), Norwalk virus, SV40, or HPV (human papilloma virus).

According to the kit, at least one protein among the fusion protein, andthe partner protein may contain a fluorescent protein in order to verifywhether the protein nano-cluster is formed. At this time, thefluorescent protein may be green fluorescent protein (GFP), yellowfluorescent protein (YFP), red fluorescent protein (RFP), orangefluorescent protein, cyan fluorescent protein (CFP), blue fluorescentprotein (BFP), far-red fluorescent protein or tetracysteine fluorescentmotif.

According to the kit, the light-induced heterodimerized protein may beCIB, CIBN, PhyB, PIF, FKF1, GIGANTEA, CRY, or PHR, and the partnerprotein may be CRY or PHR when the light-induced heterodimerized proteinis CIB or CIBN, the partner protein may be PIF when the light-inducedheterodimerized protein is PhyB, the partner protein may be GIFANTEAwhen the light-induced heterodimerized protein is FKF1, the partnerprotein may be CIB or CIBN when the light-induced heterodimerizedprotein is CRY or PHR, the partner protein may be PIF when thelight-induced heterodimerized protein is PhyB, or the partner proteinmay be FKF1 when the light-induced heterodimerized protein is GIGANTEA.The PIF may be PIF3 or PIF6.

According to the kit, the light-induced heterodimerized protein or thepartner protein may be homodimerized regardless of light irradiation. Inthis case, the light-induced heterodimerized protein or the partnerprotein capable of forming homodimer regardless of light irradiation maybe CRY or PHR.

Effect of the Invention

According to exemplary embodiments of the present invention, asdescribed above, it is possible to analyze interaction between proteinsand to control function of particular proteins reversibly andspatio-temproally by forming nanoclusters within a cell or a tissueefficiently. Of course, the scope of the present invention is notlimited thereto.

BRIEF DESCRIPTION OF DRAWINGS

FIG. 1 is a schematic overview of the principles of nano-clusterformation induced by light irradiation.

FIG. 2 is a fluorescence microscophic image showing an experimentalresult representing actual formation of nanocluster shown in FIG. 1.

FIG. 3 is a graph showing the induced nano-cluster formation anddissociation process over a period of time.

FIG. 4 is a schematic overview representing the principles of a methodfor analyzing the interaction between the proteins using the nanoclusterformation according to an embodiment of the present invention.

FIG. 5 is a schematic overview representing the principles of a methodfor analyzing the interaction between the proteins using the nanoclusterformation according to another embodiment of the present invention.

FIG. 6 is a schematic overview representing the principles of a methodfor analyzing the interaction between the proteins using the nanoclusterformation according to still another embodiment of the presentinvention.

FIG. 7 is a schematic overview representing the principles of a methodfor analyzing the interaction between the proteins using the nanoclusterformation according to still another embodiment of the presentinvention.

FIG. 8 is a schematic overview representing the principles of a methodfor analyzing the interaction between the proteins using the nanoclusterformation according to still another embodiment of the presentinvention.

FIG. 9 is a schematic overview representing the principles of a methodfor inhibiting a target protein using the nanocluster formationaccording to an embodiment of the present invention.

FIG. 10 is a schematic overview representing the principles of theinhibition of a particular protein by entrapment of the target proteininteracting with a partner protein via the formation of nanoclustersbetween the two proteins by irradiating light having a particularwavelength within partial active region of a cell.

FIG. 11 is a schematic overview representing the principles of theformation of nanoclusters within multicellular area such as a subjectand a tissue and the inhibition of a particular protein within the areaby irradiating light having a particular wavelength to area.

FIG. 12 is a schematic diagram illustrating conceptually the formationof nano-clusters and the regulation of molecular function of aparticular protein thereby by light irradiation in accordance with anembodiment of the present invention.

FIG. 13 is a schematic diagram illustrating conceptually the formationof nano-clusters by irradiating two different nanoparticles and a methodfor regulating molecular function of a particular protein therebyaccording to an embodiment of the present invention.

FIG. 14 is a schematic diagram illustrating conceptually the formationof nano-clusters by irradiating and a method for regulating molecularfunction of a particular protein thereby according to another embodimentof the present invention.

FIG. 15 is a schematic diagram illustrating conceptually the formationof nano-clusters by irradiating and a method for regulating molecularfunction of a particular protein thereby according to still anotherembodiment of the present invention.

FIG. 16 is a schematic diagram illustrating conceptually the formationof nano-clusters by irradiating and the regulation of function of PI3Kprotein using the method illustrated in FIG. 14 according to anembodiment of the present invention.

FIG. 17 is a schematic diagram illustrating conceptually the formationof nano-clusters by irradiating according to an embodiment of thepresent invention and the regulation of function of Vav2 protein usingthe same.

FIG. 18 is a fluorescent microscophic photograph representingexperimental results showing interactions between Hras and RBD raf1, andHras and RBD p110, respectively using a method for analyzing theinteraction between proteins using the nano-cluster formation accordingto an embodiment of the present invention.

FIG. 19 is a fluorescent microscophic photograph showing the result ofthe formation of nano-clusters by light irradiation and the inhibitionof a particular protein over a period of time thereby, which proves theconception illustrated in FIG. 17.

BEST MODE Definition of Terms

The terms used in this document are designated as follows.

A “bait protein” used in this document means proteins that interact withtarget proteins.

A “target protein” used in this document means a partner proteininteracting with the bait protein and may be referred as to a protein tobe analyzed.

A “self-assembled protein” or “SAP” used in this document means aprotein capable of forming an organized multimer without help ofexternal mediators and representative self-assembled protein includesferritin

A “nano-cluster” or “protein nanocluster” used in this document refersto an agglometer colonized by the interaction between the nanoparticlesformed by self-assembly of the self-assembled proteins.

A “light-induced heterodimerized protein” used in this document refersto a protein forming a heterodimer with other proteins when irradiatedwith light having a particular wavelength.

A “partner protein” used in this document refers to a target proteinforming a heterodimer with the light-induced heterodimerized protein”when irradiated with light having a particular wavelength.

A “fusion protein” used in this document refers to a protein in whichtwo or more proteins are connected each other by amino bond whilemaintaining the functionality of each unit protein.

A “heterodimer” used in this document means a dimer or a complex formedby two different proteins via the interaction between them.

A “homodimer” used in this document means a dimer or a complex formed bytwo same proteins via intermolecular interaction.

An “operably linked to” used in this document means that a particularpolynucleotide can function when connected to other polynucleotides. Inother words, “a polynucleotide encoding a particular protein, whereinthe polynucleotide is operably linked to the promoter” means that thepolynucleotide can be transcribed into mRNAs according to the action ofthe promoter and the mRNAs are translated to the protein and “apolyncleotide encoding a particular protein is operably linked to apolynucleotide encoding the other protein” means that the particularprotein can be expressed as fused to the other protein.

A “CIB” used in this document means a cryptochrome-interactingbasic-helix-loop-helix protein and a representative CIB is ArabidopsisCIB1 (GenBank No. NM_119618).

A “CIBN” used in this document means a N-terminal of the CIB, which is apart interacting with cryptochrome (CRY) when it is irradiated.

A “CRY” used in this document refers to a cryptochrome protein and arepresentative CRY is Arabidopsis CRY2 (GenBank No. NM_100320).

A “PHR” used in this document refers to an N-terminal of the CRY whichis a phytolyase homologous region of CRY and interacts with the CIB orthe CIBN when it is irradiated (Kennedy et al., Nat. Methods, 7(12):973-975, 2010)

A “Phy” used in this document refers to a phytochrome protein and arepresentative Phy is Arabidopsis PhyA (GenBank No.: NM_001123784) andthe Phy is known to interact with PIF (phytochrome interacting factor)(Min et al., Nature, 400: 781-784, 1999).

A “PIF” used in this document refers to a phytochrome interacting factorand a representative PIF includes Arabidopsis PIF1 (GenBank No.:NM_001202630), PIF3 (GenBank No.: NM_179295). PIF4 (GenBank No.:NM_180050), PIF5 (GenBank No.: NM_180690), PIF6 (GenBank No.:NM_001203231) and PIF7 (GenBank No.: NM_125520).

A “FKF” used in this document refers to a Flavin-binding, Kelch repeat,F-fox protein and a representative FKF is Arabidopsis FKF1 (GenBank No.:NM_105475). It is known to interact with GIGANTEA protein when it isirradiated (Sawa et al., Science, 318 (5848): 261-265, 2007).

A “GIGANTEA” used in this document refers to a protein related tophytochrome signal transduction and is known to regulate flowering timeof flowers.

A “tetracysteine fluorescent” used in this document refers to apolypeptide containing an amino acid sequence of Cys-Cys-Xaa-Xaa-Cys-Cys(SEQ ID NO: 1), wherein the Xaa is any one amino acid except cysteineand fluorescent pattern varies depending on the type of Xaa and thelength of the polypeptide (Adams et al., J. Am. Chem. Soc., 124:6063-6077, 2002).

Methods for analyzing protein interaction according to variousembodiments of the present invention are described through theaccompanying drawings.

FIG. 1 is a schematic overview of the principles of nano-clusterformation induced by light irradiation.

The present inventors hypothesized that a protein nano-cluster may beformed by light-induced interaction between two light-inducedheterodimerized proteins, if a light-induced heterodimerized proteinexpressed as fused to a self-assembled protein such as ferritin and apartner protein interacting with the light-induced heterodimerizedprotein by light irradiating are co-expressed and irradiated with alight having a particular wavelength capable of inducing thelight-induced interaction between two proteins (See FIG. 1). Thus, thepresent inventors co-transfected the cell with an expression vectorcontaining a gene construct encoding a fusion protein ferritin which isa self-assembled protein, CIBN which is a light-induced heterodimerizedprotein, and a fluorescent protein and an expression vector containing agene construct encoding PHR which is a partner protein and irradiatedthe co-transfected cell with a light having wavelength inducing theinteraction between the CIBN and the PHR observed sopts emitting strongfluorescent within a cell in order to verify the hypothesis, and thenthe present inventors observed sopts emitting strong fluorescent withina cell (FIG. 2) and confirmed that waxes and wanes of number of spotscoincided with light irradiation cycle (FIG. 3). This result proves thata nano-cluster between self-assembled fusion proteins within a cell wasformed by light irradiation.

In an aspect to the present invention, an expression vector comprising apolynucleotide encoding a fusion protein comprising a light-inducedheterodimerzable protein, and a self-assembled protein is provided.

According to the expression vector, the light-induced heterodimerizedprotein may be CIB, CIBN PhyB, PIF FKF1, GIGANTEA, CRY, or PHR.

According to the expression vector, the self-assembled protein may beferritin, virus capsid protein, ferritin-like protein,calcium/calmodulin-dependent protein kinase II alpha subunit (CaMKIIα)or DsRed, and the virus capsid protein may be a capsid protein derivedfrom CCMV (cowpea chlorotic mottle virus), Norwalk virus, SV40, or HPV(human papilloma virus). The description of the self-assembled protein,will be applied to a first self-assembled protein and a secondself-assembled protein in the same manner which are described later.

According to the expression vector, the fusion protein may furthercontain a fluorescent protein, and the fluorescent protein may be addedto N-terminal or C-terminal of the fusion protein and may be insertedbetween the light-induced heterodimerized protein and the self-assembledprotein. In this case, the fluorescent protein may be green fluorescentprotein (GFP), yellow fluorescent protein (YFP), red fluorescent protein(RFP), orange fluorescent protein, cyan fluorescent protein (CFP), bluefluorescent protein (BFP), or tetracysteine fluorescent motif. The greenfluorescent protein may be EGFP (enhanced green fluorescent protein),Emerald (Tsien, Annu. Rev. Biochem., 67: 509-544, 1998), Superfolder(Pedelacq et al., Nat. Biotech., 24: 79-88, 2006), GFP (Prendergast etal., Biochem., 17 (17): 3448-3453, 1978), Azami Green (Karasawa, et al.,J. Biol. Chem., 278: 34167-34171, 2003), TagGFP (Evrogen, Russia),TurboGFP (Shagin et al., Mol. Biol. Evol., 21 (5): 841-850, 2004),ZsGreen (Matz et al., Nat. Biotechnol., 17: 969-973, 1999) or T-Sapphire(Zapata-Hommer et al, BMC Biotechnol., 3:5, 2003). The yellowfluorescent protein may be EYFP (enhanced yellow fluorescent protein,Tsien, Annu. Rev. Biochem., 67: 509-544, 1998), Topaz (Hat et al., Ann.NY Acad. Sci., 1: 627-633, 2002), Venus (Nagai et al., Nat. Biotechnol.,20(1): 87-90, 2002), mCitrine (Griesbeck et al., J. Biol. Chem., 276:29188-29194, 2001), Ypet (Nguyet and Daugherty, Nat. Biotechnol., 23(3):355-360, 2005), TagYFP (Evrogen, Russia), PhiYFP (Shagin et al., Mol.Biol. Evol., 21(5): 841-850, 2004), ZsYellow1 (Matz et al., Nat.Biotechnol., 17: 969-973, 1999), or mBanana (Shaner et al., Nat.Biotechnol., 22: 1567-1572, 2004). The red fluorescent protein may bemRuby (Kredel et al., PLoS ONE, 4(2): e4391, 2009), mApple (Shaner etal., Nat. Methods, 5(6): 545-551, 2008), mStrawberry (Shaner et al.,Nat. Biotechnol., 22: 1567-1572, 2004) and AsRed2 (Shanner et al., Nat.Biotechnol., 22: 1567-1572, 2004) or mRFP (Campbell et al., Proc. Natl.Acad. Sci. USA, 99(12): 7877-7882, 2002). The orange fluorescent proteinmay be Kusabira Orange (Karawawa et al., Biochem. J. 381(Pt 1): 307-312,2004), Kusabira Orange2 (MBL International Corp., Japan), mOrange(Shaner et al., Nat. Biotechnol., 22: 1567-1572, 2004), mOrange2 (Shaneret al., Nat. Biotechnol., 22: 1567-1572, 2004), dTomato (Shaner et al.,Nat. Biotechnol., 22: 1567-1572, 2004), dTomato-Tandem (Shaner et al.,Nat. Biotechnol., 22: 1567-1572, 2004), TagRFP (Merzlyak et al., Nat.Methods, 4(7): 555-557, 2007), TagRFP-T (Shaner et al., Nat. Methods,5(6): 545-551, 2008), DsRed (Baird et al., Proc. Natl. Acad. Sci. USA,97: 11984-11989, 1999), DsRed2 (Clontech, USA), DsRed-Express (Clontech,USA), DsRed-Monomer (Clontech, USA), or mTangerine (Shaner et al., NatBiotechnol, 22: 1567-1572, 2004 above). The cyan fluorescent protein maybe ECFP (enhanced cyan fluorescent protein, Cubitt et al., TrendsBiochem. Sci., 20: 448-455, 1995), mECFP (Ai et al., Biochem. J.,400(3): 531-540, 2006), mCerulean (Koushik et al., Biophys. J., 91(12):L99-L101, 2006), CyPet (Nguyet and Daugherty, Nat. Biotechnol., 23 (3):355-360, 2005), AmCyan1 (Matz et al., Nat. Biotechnol., 17: 969-973,1999), Midori-Ishi Cyan (Karawawa et al., Biochem. J., 381(Pt 1):307-312, 2004), TagCFP (Evrogen, Russia) or mTFP1, (Ai et al, Biochem.J., 400 (3): 531-540, 2006). The blue fluorescent protein may be EBFP(enhanced blue fluorescent protein, Clontech, USA), EBFP2 (Ai et al.,Biochemistry, 46 (20): 5904-5910. 2007), Azurite (Mena et al., Nat.Biotechnol., 24: 1569-1571, 2006) or mTagBFP (Subach et al., Chem.Biol., 15(10): 1116-1124, 2008). The far red fluorescent protein may bemPlum (Wang et al., Proc. Natl. Acad. Sci. USA, 101: 16745-16749, 2004),mCherry (Shanner et al., Nat. Biotechnol., 22: 1567-1572, 2004),dKeima-Tandem (Kogure et al., Methods, 45(3): 223-226, 2008), JRed(Shagin et al., Mol. Biol. Evol., 21(5): 841-850, 2004), mRaspberry(Shanner et al., Nat. Biotechnol., 22: 1567-1572, 2004), HcRed1 (Fradkovet al., Biochem. J., 368(Pt 1): 17-21, 2002), HcRed-Tandem (Fradkov etal., Nat. Biotechnol., 22(3): 289-296, 2004), AQ143 (Shkrob et al.,Biochem. J., 392: 649-654, 2005). The tetracysteine fluorescent motifmay be a polypeptide including an amino acid sequence ofCys-Cys-Xaa-Xaa-Cys-Cys (SEQ ID NO: 1), wherein the Xaa is any one aminoacid except cysteine. The description for the fluorescent proteinsapplied to a first fluorescent protein, a second fluorescent proteins, athird fluorescent protein and a fourth fluorescent protein which will bedescribed later in a same manner.

In an aspect to the present invention, a kit for forming light-inducedprotein nano-clusters comprising a first expression vector including apolynucleotide encoding a first fusion protein comprising alight-induced heterodimerized protein and a first self-assembledprotein; and

a second expression vector including a polynucleotide encoding a partnerprotein capable of forming a heterodimer with the light-inducedheterodimerizing protein, or a second fusion protein containing thepartner protein and a second self-assembled protein is provided.

According to the kit, the light-induced heterodimerized protein may beCIB, CIBN, PhyB, PIF, FKF1, GIGANTEA, CRY, or PHR.

According to the kit, the partner protein may be CRY or PHR when thelight-induced heterodimerized protein is CIB or CIBN, the partnerprotein may be PIF when the light-induced heterodimerized protein isPhyB, the partner protein may be GIFANTEA when the light-inducedheterodimerized protein is FKF1, the partner protein may be CIB, CIBN,PhyB, PIF, FKF1, GIGANTEA, CRY, or PHR, and the partner protein may beCRY or PHR when the light-induced heterodimerized protein is CIB orCIBN, the partner protein may be PIF when the light-inducedheterodimerzed protein is PhyB, the partner protein may be GIFANTEA whenthe light-induced heterodimerized protein is FKF1, the partner proteinmay be CIB or CIBN when the light-induced heterodimerized protein is CRYor PHR, the partner protein may be PIF when the light-inducedheterodimerized protein is PhyB, or the partner protein may be FKF1 whenthe light-induced heterodimerzed protein is GIGANTEA. The PIF may bePIF3 or PIF6. Among the light-induced heterodimerized protein or thepartner protein, CRY or PHR may be homodimerized regardless of lightirradiation.

According to the kit, the first self-assembled protein and the secondself-assembled protein may be independently ferritin, virus capsidprotein, ferritin-like protein, calcium/calmodulin-dependent proteinkinase II alpha subunit (CaMKIIα) or DsRed and the virus capsid proteinmay be a capsid protein derived from CCMV (cowpea chlorotic mottlevirus), Norwalk virus, SV40, or HPV (human papilloma virus). However,the first self-assembled protein and the second self-assembling proteinmay be same or different when they are used at the same time.

According to the kit, at least one among the first fusion protein, thepartner protein and the second fusion protein may further comprises afluorescent protein and the fluorescent protein may be added toN-termius or C-terminus of the first fusion protein, the partner proteinand/or the second fusion protein or may be inserted between thelight-induced heterodimerized protein and the first self-assembledprotein or between the partner protein and the second self-assembledprotein. At this time, the fluorescent protein may be green fluorescentprotein (GFP), yellow fluorescent protein (YFP), red fluorescent protein(RFP), orange fluorescent protein, cyan fluorescent protein (CFP), bluefluorescent protein (BFP), or tetracysteine fluorescent motif. The greenfluorescent protein may be EGFP, Emerald, Superfolder GFP, Azami Green,TagGFP, TurboGFP, ZsGreen or T-Sapphire. The yellow fluorescent proteinmay be EYFP, Topaz, Venus, mCitrine, Ypet, TagYFP, PhiYFP, mBanana, orZsYellow1. The red fluorescent protein may be mRuby, mApple,mStrawberry, AsRed2 or mRFP. The orange fluorescent protein may beKusabira Orange, Kusabira Orange2, mOrange, mOrange2, dTomato,dTomato-Tandem, TagRFP, TagRFP-T, DsRed, DsRed2, DsRed-Express,DsRed-Monomer or mTangerine. The cyan fluorescent protein may be ECFP,mECFP, mCerulean, CyPet, AmCyan1, Midori-Ishi Cyan, TagCFP, or mTFP1.The blue fluorescent protein may be EBFP, EBFP2, Azurite or mTagBFP. Thefar red fluorescent protein may be mPlum, mCherry, the dKeima-Tandem,JRed, mRaspberry, HcRed1, HcRed-Tandem, or AQ143. The tetracysteinefluorescent motif may be a polypeptide including an amino acid sequenceof Cys-Cys-Xaa-Xaa-Xaa-Cys-Cys (SEQ ID NO: 1), wherein the Xaa is one ofany amino acids except cysteine.

In another aspect to the present invention, a method for forming aprotein nano-cluster is provided, wherein the method comprises:providing a first expression vector including a polynucleotide encodinga first fusion protein comprising a light-induced heterodimerizedprotein and a first self-assembled protein; and a second expressionvector including a polynucleotide encoding a partner protein capable offorming a heterodimer with the light-induced heterodimerizing protein,or a second fusion protein containing the partner protein and a secondself-assembled protein;

transforming a cell, a tissue or a subject with the first expressionvector and the second expression vector; and

irradiating light having wavelength capable of indcucingheterodimerization between the light-induced heterodimerized protein andthe partner protein to the cell, the tissue or the subject.

According to the method, the light-induced heterodimerized protein maybe CIB, CIBN, PhyB, PIF, FKF1, GIGANTEA, CRY, or PHR.

According to the method, the partner protein may be CRY or PHR when thelight-induced heterodimerized protein is CIB or CIBN, the partnerprotein may be PIF when the light-induced heterodimerized protein isPhyB, the partner protein may be GIFANTEA when the light-inducedheterodimerized protein is FKF1, the partner protein may be CIB, CIBN,PhyB, PIF, FKF1, GIGANTEA, CRY, or PHR, and the partner protein may beCRY or PHR when the light-induced heterodimerized protein is CIB orCIBN, the partner protein may be PIF when the light-inducedheterodimerized protein is PhyB, the partner protein may be GIFANTEAwhen the light-induced heterodimerized protein is FKF1, the partnerprotein may be CIB or CIBN when the light-induced heterodimerizedprotein is CRY or PHR, the partner protein may be PIF when thelight-induced heterodimerized protein is PhyB, or the partner proteinmay be FKF1 when the light-induced heterodimerized protein is GIGANTEA.The PIF may be PIF3 or PIF6. Among the light-induced heterodimerizedprotein or the partner protein, CRY or PHR may be homodimerizedregardless of light irradiation.

According to the method, the first self-assembled protein and the secondself-assembled protein may be independently ferritin, virus capsidprotein, ferritin-like protein, calcium/calmodulin-dependent proteinkinase II alpha subunit (CaMKIIα) or DsRed and the virus capsid proteinmay be a capsid protein derived from CCMV (cowpea chlorotic mottlevirus), Norwalk virus, SV40, or HPV (human papilloma virus). However,the first self-assembled protein and the second self-assembling proteinmay be same or different when they are used at the same time.

According to the method, at least one among the first fusion protein,the partner protein and the second fusion protein may further comprisesa fluorescent protein and the fluorescent protein may be added toN-termius or C-terminus of the first fusion protein, the partner proteinand/or the second fusion protein or may be inserted between thelight-induced heterodimerized protein and the first self-assembledprotein or between the partner protein and the second self-assembledprotein. In this case, the fluorescent protein may be green fluorescentprotein (GFP), yellow fluorescent protein (YFP), red fluorescent protein(RFP), orange fluorescent protein, cyan fluorescent protein (CFP), bluefluorescent protein (BFP), or tetracysteine fluorescent motif. The greenfluorescent protein may be EGFP, Emerald, Superfolder GFP, Azami Green,TagGFP, TurboGFP, ZsGreen or T-Sapphire. The yellow fluorescent proteinmay be EYFP, Topaz, Venus, mCitrine, Ypet, TagYFP, PhiYFP, mBanana, orZsYellow1. The red fluorescent protein may be mRuby, mApple,mStrawberry, AsRed2 or mRFP. The orange fluorescent protein may beKusabira Orange, Kusabira Orange2, mOrange, mOrange2, dTomato,dTomato-Tandem, TagRFP, TagRFP-T, DsRed, DsRed2, DsRed-Express,DsRed-Monomer or mTangerine. The cyan fluorescent protein may be ECFP,mECFP, mCerulean, CyPet, AmCyan1, Midori-Ishi Cyan, TagCFP, or mTFP1.The blue fluorescent protein may be EBFP, EBFP2, Azurite or mTagBFP. Thefar red fluorescent protein may be mPlum, mCherry, the dKeima-Tandem,JRed, mRaspberry, HcRed1, HcRed-Tandem, or AQ143. The tetracysteinefluorescent motif may be a polypeptide including an amino acid sequenceof Cys-Cys-Xaa-Xaa-Xaa-Cys-Cys (SEQ ID NO: 1), wherein the Xaa is one ofany amino acids except cysteine.

The method and kit for forming a protein nano-cluster according to anembodiment of the present invention may be useful for screening asubstance regulating protein-protein interaction as well as analyzingprotein-protein interaction by inducing the formation of a nano-clusterbetween nanoparticles formed by protein self-assembly by lightirradiation. In addition, the method and kit for forming a proteinnano-cluster according to an embodiment of the present invention may beuseful for analyzing real-time protein-protein interaction within livingcells with minimal cell damage, because it can induce protein-proteininteraction reversibly. Moreover, the method and kit for forming aprotein nano-cluster according to an embodiment of the present inventionmay be useful for inhibiting a target protein reversibly by entrappingthe target protein using a bait protein interacting the target protein.

The aforementioned method for analyzing protein-protein interaction andthe method for inhibiting a target protein reversibly are described indetail as follows:

In an aspect of the present invention, a kit for analyzing interactionbetween proteins is provided, wherein the kit comprises: a firstexpression vector including a first gene construct containing a promoterand a first polynucleotide encoding a first fusion protein comprising abait protein and a first self-assembled protein, wherein the firstpolynucleotide is operably linked to the promoter: a second expressionvector including a second gene construct containing a promoter and asecond polynucleotide encoding a second fusion protein comprising alight-induced heterodimerized protein and the first self-assembledprotein, wherein the second polynucleotide is operably linked to thepromoter; a third expression vector including a third gene constructcontaining a promoter and a third polynucleotide encoding a third fusionprotein comprising a partner protein interacting with the light-inducedheterodimerized protein, a second fluorescent protein, and a secondself-assembled protein, wherein the third polynucleotide is operablylinked to the promoter; and a fourth expression vector including afourth gene construct containing a promoter and a fourth polynucleotideencoding a fourth fusion protein comprising a target protein and a thirdfluorescent protein, wherein the fourth polynucleotide is operablylinked to the promoter, with the proviso that at least one fusionprotein among the first fusion protein and the second fusion proteincontains a first fluorescent protein, wherein the first self-assembledprotein and the second self-assembled protein do not interact eachother, and wherein all the fluorescent proteins emit differentwavelengths of light, wherein the first self-assembled protein or thesecond self-assembled protein may be omitted from any fusion proteinscontaining DsRed when either the first fluorescent protein or the secondfluorescent protein is DsRed. (FIG. 4).

According to the kit, the order of the bait protein, the firstself-assembled protein and optionally the first fluorescent proteinwithin the first fusion protein may be rearranged. For example, thefirst fusion protein may consist of the bait protein, the firstfluorescent protein and the first assemblem protein in consecutive orderor may consist of the first self-assembled protein and the firstfluorescent protein and the bait protein in consecutive order.Similarly, the order of the light-induced heterodimerized protein, thefirst self-assembled protein and the optional first fluorescent proteinwithin the second fusion protein may be rearranged as necessary. Forexample, the second fusion protein may consist of the light-inducedheterodimerized protein, the first fluorescent protein and the firstself-assembled protein in consecutive order or may consist of the firstself-assembled protein, the first fluorescent protein and thelight-induced heterodimerized protein in consecutive order. Similarly,the order of the partner protein, the second fluorescent protein and thesecond self-assembled protein within the third fusion protein may beadjusted as necessary. For example, the third fusion protein may consistof the partner protein, the second fluorescent protein and the secondself-assembled protein in consecutive order or may consist of the secondself-assembled protein and the partner protein, in consecutive order.Finally, in the fourth fusion protein, the target protein may bepositioned at N-terminal or the third fluorescent protein may bepositioned at the N-terminal.

The all fusion proteins described above may contain at least one linkerbetween the unit proteins consisting each fusion protein.

In another aspect of the present invention, a kit for analyzinginteraction between proteins is provided, wherein the kit comprises: afirst expression vector including a first gene construct containing apromoter, a first polynucleotide encoding a first fusion proteincomprising a first self-assembled protein and a multicloning site inwhich a polynucleotide encoding a bait protein is operably linked to thefirst polynucleotide and wherein the first polynucleotide is operablylinked to the promoter; a second expression vector including a secondgene construct containing a promoter, and a second polynucleotideencoding a second fusion protein comprising a light-inducedheterodimerized protein, wherein the second polynucleotide is operablylinked to the promoter; a third expression vector including a third geneconstruct containing a promoter and a third polynucleotide encoding athird fusion protein comprising a partner protein interacting with thelight-induced heterodimerized protein, a second fluorescent protein, anda second self-assembled protein, wherein the third polynucleotide isoperably linked to the promoter; and a fourth expression vectorincluding a fourth gene construct containing a promoter and a fourthpolynucleotide encoding a fourth fusion protein comprising a thirdfluorescent protein and a multicloning site in which a polynucleotideencoding a target protein is operably linked to the fourthpolynucleotide and wherein the fourth polynucleotide is operably linkedto the promoter, with the proviso that at least one fusion protein amongthe first fusion protein and the second fusion protein contains a firstfluorescent protein, wherein the first self-assembled protein and thesecond self-assembled protein do not interact each other, and whereinall the fluorescent proteins emit different wavelengths of light,wherein the first self-assembled protein or the second self-assembledprotein may be omitted from any fusion proteins containing DsRed wheneither the first fluorescent protein or the second fluorescent proteinis DsRed.

According to the kit, the first to third fluorescent proteins may begreen fluorescent protein (GFP), yellow fluorescent protein (YFP), redfluorescent protein (RFP), orange fluorescent protein, cyan fluorescentprotein (CFP), blue fluorescent protein (BFP), TagCFP, DsRed ortetracysteine fluorescent motif, independently.

According to the kit, the first self-assembled protein and the secondself-assembled protein may be independently ferritin, virus capsidprotein, ferritin-like protein, calcium/calmodulin-dependent proteinkinase II alpha subunit (CaMKIIα) or DsRed and the virus capsid proteinmay be a capsid protein derived from CCMV (cowpea chlorotic mottlevirus), Norwalk virus, SV40, or HPV (human papilloma virus). However,the first self-assembled protein and the second self-assembling proteinmay be same or different when they are used at the same time.

According to the kit, the light-induced heterodimerized protein may beCIB, CIBN, PhyB, PIF, FKF1, GIGANTEA, CRY, or PHR.

According to the kit, the partner protein may be CRY or PHR when thelight-induced heterodimerized protein is CIB or CIBN, the partnerprotein may be PIF when the light-induced heterodimerized protein isPhyB, the partner protein may be GIFANTEA when the light-inducedheterodimerized protein is FKF1, the partner protein may be CIB, CIBN,PhyB, PIF, FKF1, GIGANTEA, CRY, or PHR, and the partner protein may beCRY or PHR when the light-induced heterodimerized protein is CIB orCIBN, the partner protein may be PIF when the light-inducedheterodimerized protein is PhyB, the partner protein may be GIFANTEAwhen the light-induced heterodimerized protein is FKF1, the partnerprotein may be CIB or CIBN when the light-induced heterodimerizedprotein is CRY or PHR, the partner protein may be PIF when thelight-induced heterodimerized protein is PhyB, or the partner proteinmay be FKF1 when the light-induced heterodimerized protein is GIGANTEA.The PIF may be PIF3 or PIF6. Among the light-induced heterodimerizedprotein or the partner protein, CRY or PHR may be homodimerizedregardless of light irradiation.

According to the kit, the promoter may be a eukaryotic promoter.

FIG. 4. is a schematic overview representing the principles of a methodfor analyzing the interaction between the proteins using the nanoclusterformation according to an embodiment of the present invention. As shownin FIG. 4, the light-induced nano-cluster may be formed by thelight-induced interaction between a first nanoparticle including alight-induced heterodimerized protein (i.e., CIBN) and a secondnanoparticle including a partner protein interacting with thelight-induced heterodimerized protein (i.e., PHR).

In this case, the first nanoparticle is formed by self-assembly of twofusion protein (a first fusion protein and a second fusion protein)expressed by two gene construct, respectively and the first fusionprotein and the second fusion protein have a common self-assembledprotein (the first self-assembled protein, 1^(st) SAP), thus ahetero-assembled nanoparticle is formed. In order to check theexpression of the fusion proteins and the formation of the nanocluster,the first fusion protein and/or the second fusion protein contain afluorescent protein. On the other hand, the second nanoparticle is ahomo-assembled nanoparticle formed by self assembly of a third fusionprotein comprising a second self-assembled protein (2^(nd) SAP) notinteracting with the first self-assembled protein and a partner proteininteracting with the light-induced heterodimerized protein by lightirradiation. The third fusion protein contains a second fluorescentprotein emitting different wavelengths of light to the first fluorescentprotein in order to check the expression of the fusion protein and theformation of nanoclusters. On the other hand, the target protein to beexamined whether it interacts with the bait protein is expressed as afusion protein with a third fluorescent protein emitting differentwavelengths of light to the first and second fluorescent protein. If thetarget protein interacts with the bait protein, the nano-cluster formedby the light-induced interaction between the light-inducedheterodimerized protein and the partner protein and the target proteinwill be co-localized. However, if the target protein does not interactwith the bait protein, fluorescent corresponding the target protein willbe dispersed throughout cytoplasm of a cell rather than forming strongfluorescent spots which are formed by the first nanopartides and thesecond nanoparticles. On the other hand, nano-clusters formed by thehomo-interaction between the first nanoparticles are distinguished fromthe nano-clusters formed by the hetero-interaction, since thefluorescent pattern formed by the first nanoparticles does notcorrespond to that formed by the second nanoparticles.

The CIBN illustrated in FIG. 4 refers to an N-terminal fragment (1-170a.a.) of A. thaliana bHLH63 protein. It is a representativelight-induced heterodimerized protein known to interact with PHR(phytolyase homologous region) which is an N-terminal fragment (1-488a.a.) of A. thaliana cryptochrome 2 when it is irradiated with bluelight having 488 nm of wavelength (Liu et al., Science, 322(5907):1535-1539, 2009). Hereinafter, the light-induced heterodimerized proteinwill be designated as CIBN and the partner protein will be referred toCRY2 or PHR, for convenience. The light-induced heterodimerized proteinmay be PhyB, PIF3, PIF6, FKF1, or GIGANTEA, etc. besides CIB and CIBN.The PhyB is A. thaliana phytochrome B protein and known to interact withPIF (phytochrome-interacting factor) when irradiated with red light(Castillon et al., Trends Plant Sci., 12(11): 514-521, 2007). FKF1(flavin-binding, kelch repat, F-box 1) is a protein regulated by timewhich is known to regulate flowering time and interacts with GIGANTEAwhen irradiated with blue light (Sawa et al., Science, 318(5848):261-265, 2007). GIGANTEA is a nuclear protein involved in phytochromesignal transduction in A. thaliana (Huq et al., Proc. Natl. Acad. Soc.USA, 97(17): 9789-9794, 2000).

In another aspect of the present invention, a kit for analyzinginteraction between proteins is provided, wherein the kit comprises: afirst expression vector including a first gene construct containing apromoter and a first polynucleotide encoding a first fusion proteincomprising a light-induced heterodimerized protein, a first fluorescentprotein and a first self-assembled protein, wherein the firstpolynucleotide is operably linked to the promoter; a second expressionvector including a second gene construct containing a promoter and asecond polynucleotide encoding a second fusion protein comprising apartner protein interacting with the light-induced heterodimerizedprotein, a second fluorescent protein and a bait protein, wherein thesecond polynucleotide is operably linked to the promoter; and a thirdexpression vector including a third gene construct containing a promoterand polynucleotide encoding a third fusion protein comprising a targetprotein, a third fluorescent protein and a second self-assembledprotein, wherein the third polynucleotide is operably linked to thepromoter, with the proviso that all the fluorescent proteins emitdifferent wavelengths of light, wherein the first self-assembled proteinand the second self-assembled protein do not interact each other, andwherein the first self-assembled protein or the second self-assembledprotein may be omitted from any fusion proteins containing DsRed wheneither the first fluorescent protein or the third fluorescent protein isDsRed (FIG. 5).

In another aspect of the present invention, a kit for analyzinginteraction between proteins is provided, wherein the kit comprises: afirst expression vector including a first gene construct containing apromoter and a first polynucleotide encoding a first fusion proteincomprising a light-induced heterodimerized protein, a first fluorescentprotein and a first self-assembled protein, wherein the firstpolynucleotide is operably linked to the promoter; a second expressionvector including a second gene construct containing a promoter and asecond polynucleotide encoding a second fusion protein comprising apartner protein interacting with the light-induced heterodimerizedprotein and a second fluorescent protein and a multicloning site inwhich a polynucleotide encoding a bait protein is operably linked to thesecond polynucleotide, wherein the second polynucleotide is operablylinked to the promoter; and a third expression vector including a thirdgene construct containing a promoter and a third polynucleotide encodinga third fusion protein comprising a third fluorescent protein and asecond self-assembled protein and a multicloning site in which apolynucleotide encoding a target protein is operably linked to the thirdpolynucleotide, wherein the third polynucleotide is operably linked tothe promoter, with the proviso that all the fluorescent proteins emitdifferent wavelengths of light, wherein the first self-assembled proteinand the second self-assembled protein do not interact each other, andwherein the first self-assembled protein or the second self-assembledprotein may be omitted from any fusion proteins containing DsRed wheneither the first fluorescent protein or the third fluorescent protein isDsRed.

According to the kit, the first to third fluorescent proteins may begreen fluorescent protein (GFP), yellow fluorescent protein (YFP), redfluorescent protein (RFP), orange fluorescent protein, cyan fluorescentprotein (CFP), blue fluorescent protein (BFP), TagCFP, DsRed ortetracysteine fluorescent motif, independently.

According to the kit, the first self-assembled protein and the secondself-assembled protein may be independently ferritin, virus capsidprotein, ferritin-like protein, calcium/calmodulin-dependent proteinkinase II alpha subunit (CaMKIIα) or DsRed and the virus capsid proteinmay be a capsid protein derived from CCMV (cowpea chlorotic mottlevirus), Norwalk virus, SV40, or HPV (human papilloma virus). However,the first self-assembled protein and the second self-assembling proteinmay be same or different when they are used at the same time.

According to the kit, the light-induced heterodimerized protein may beCIB, CIBN, PhyB, PIF, FKF1, GIGANTEA, CRY, or PHR.

According to the kit, the partner protein may be CRY or PHR when thelight-induced heterodimerized protein is CIB or CIBN, the partnerprotein may be PIF when the light-induced heterodimerized protein isPhyB, the partner protein may be GIFANTEA when the light-inducedheterodimerized protein is FKF1, the partner protein may be CIB, CIBN,PhyB, PIF, FKF1, GIGANTEA, CRY, or PHR, and the partner protein may beCRY or PHR when the light-induced heterodimerized protein is CIB orCIBN, the partner protein may be PIF when the light-inducedheterodimerized protein is PhyB, the partner protein may be GIFANTEAwhen the light-induced heterodimerized protein is FKF1, the partnerprotein may be CIB or CIBN when the light-induced heterodimerizedprotein is CRY or PHR, the partner protein may be PIF when thelight-induced heterodimerized protein is PhyB, or the partner proteinmay be FKF1 when the light-induced heterodimerized protein is GIGANTEA.The PIF may be PIF3 or PIF6.

According to the kit, the promoter may be a eukaryotic promoter.

FIG. 5 is a schematic overview representing the principles of a methodfor analyzing the interaction between the proteins using the nanoclusterformation according to another embodiment of the present invention. Asshown in FIG. 5, nano-clusters are not generated directly by theinteraction between nanoparticles formed by self-assembly rather formedindirectly by a cluster formation linker. Particularly, a firstnanoparticle is generated by self-assembly of the first fusion proteincontaining the light-induced heterodimerized protein, the firstfluorescent protein and the first self-assembled protein (1^(st) SAP). Asecond nanoparticle is formed by self-assembly of the third fusionprotein containing the target protein and the third fluorescent proteinemitting different wavelengths of light to the first fluorescent proteinand the second self-assembled protein. When a second fusion proteincontaining the partner protein besides the first nanoparticle and thesecond nanoparticle, the second fluorescent protein and the bait proteinis co-expressed within a cell, a heterodimer is formed between thelight-induced heterodimerized protein and the partner protein and if thetarget protein displayed onto the surface of the second nanoparticleinteracts with the bait protein a nanocluster is generated by thelinkage of the second fusion protein and it is possible to determinewhether a nano-cluster is generated and it is false-positive orfalse-negative through unique fluorescent patterns of each fusionprotein. If the target protein does not interact with the bait protein,no nano-cluster is generated. On the contrary, if nano-clusters areformed by homodimerization of any one among the light-inducedheterodimerized protein, the partner protein, the bait protein and thetarget protein, one can distinguish false-positive and false-negativethrough different fluorescent patterns according to each fluorescentprotein.

In another aspect of the present invention, a kit for analyzinginteraction between proteins is provided, wherein the kit comprises: afirst expression vector including a first gene construct containing apromoter, a first polynucleotide encoding a first fusion proteincomprising a bait protein and a self-assembled protein, wherein thefirst polynucleotide is operably linked to the promoter; a secondexpression vector including a second gene construct containing apromoter, and a second polynucleotide encoding a second fusion proteincomprising a light-induced heterodimerized protein and the selfassembled protein, wherein the second polynucleotide is operably linkedto the promoter; a third expression vector including a third geneconstruct containing a promoter and a third polynucleotide encoding athird fusion protein comprising at least two repeated partner proteinsinteracting with the light-induced heterodimerized protein, a secondfluorescent protein, wherein the third polynucleotide is operably linkedto the promoter; and a fourth expression vector including a fourth geneconstruct containing a promoter and a fourth polynucleotide encoding afourth fusion protein comprising a third fluorescent protein wherein thefourth polynucleotide is operably linked to the promoter, with theproviso that at least one fusion protein among the first fusion proteinand the second fusion protein contains a first fluorescent protein,wherein all fluorescent proteins emit different wavelengths of light andwherein the first self-assembled protein or the second self-assembledprotein may be omitted from the first fusion protein or the secondfusion protein when the first fluorescent protein protein is DsRed (FIG.6).

In another aspect of the present invention, a kit for analyzinginteraction between proteins is provided, wherein the kit comprises: afirst expression vector including a first gene construct containing apromoter, a first polynucleotide encoding a first fusion proteincontaining self-assembled protein and a multicloning site in which apolynucleotide encoding a bait protein is operably linked to the firstpolynucleotide and wherein the first polynucleotide is operably linkedto the promoter; a second expression vector including a second geneconstruct containing a promoter, and a second polynucleotide encoding asecond fusion protein comprising a light-induced heterodimerized proteinand the self assembled protein, wherein the second polynucleotide isoperably linked to the promoter; a third expression vector including athird gene construct containing a promoter and a third polynucleotideencoding a third fusion protein comprising at least two repeated partnerproteins interacting with the light-induced heterodimerized protein, asecond fluorescent protein, wherein the third polynucleotide is operablylinked to the promoter; and a fourth expression vector including afourth gene construct containing a promoter and a fourth polynucleotideencoding a fourth fusion protein comprising a third fluorescent proteinwherein the fourth polynucleotide is operably linked to the promoter,with the proviso that at least one fusion protein among the first fusionprotein and the second fusion protein contains a first fluorescentprotein, wherein all the fluorescent proteins emit different wavelengthsof light, and wherein the first self-assembled protein or the secondself-assembled protein may be omitted from the first fusion protein orthe second fusion protein when the first fluorescent protein is DsRed.

According to the kit, the first to third fluorescent proteins may begreen fluorescent protein (GFP), yellow fluorescent protein (YFP), redfluorescent protein (RFP), orange fluorescent protein, cyan fluorescentprotein (CFP), blue fluorescent protein (BFP), TagCFP, DsRed ortetracysteine fluorescent motif, independently.

According to the kit, the light-induced heterodimerized protein may beCIB, CIBN, PhyB, PIF, FKF1, GIGANTEA, CRY, or PHR.

According to the kit, the partner protein may be CRY or PHR when thelight-induced heterodimerized protein is CIB or CIBN, the partnerprotein may be PIF when the light-induced heterodimerized protein isPhyB, the partner protein may be GIFANTEA when the light-inducedheterodimerized protein is FKF1, the partner protein may be CIB, CIBN,PhyB, PIF, FKF1, GIGANTEA, CRY, or PHR, and the partner protein may beCRY or PHR when the light-induced heterodimerized protein is CIB orCIBN, the partner protein may be PIF when the light-inducedheterodimerized protein is PhyB, the partner protein may be GIFANTEAwhen the light-induced heterodimerized protein is FKF1, the partnerprotein may be CIB or CIBN when the light-induced heterodimerizedprotein is CRY or PHR, the partner protein may be PIF when thelight-induced heterodimerized protein is PhyB, or the partner proteinmay be FKF1 when the light-induced heterodimerized protein is GIGANTEA.The PIF may be PIF3 or PIF6.

According to the kit, the self-assembled protein may be ferritin, viruscapsid protein, ferritin-like protein, calcium/calmodulin-dependentprotein kinase II alpha subunit (CaMKIIα) or DsRed and the virus capsidprotein may be a capsid protein derived from CCMV (cowpea chloroticmottle virus), Norwalk virus, SV40, or HPV (human papilloma virus).

According to the kit, the promoter may be a eukaryotic promoter.

FIG. 6 is a schematic overview representing the principles of a methodfor analyzing the interaction between the proteins using the nanoclusterformation according to still another embodiment of the presentinvention. As shown in FIG. 6, according to the example, a singlenanoparticle is used. The nanoparticle forms a hetero-assemblednanoparticle between a first fusion protein comprising a light-inducedheterodimerized protein and a self-assembled protein, and a secondfusion protein comprising a bait protein and the self-assemble protein.In this case, the first fluorescent protein is incorporated to eitherthe first fusion protein or the second fusion protein in order todetermine whether the fusion proteins are expressed and nano-clustersare generated. The formation of nano-clusters is accomplished by thelight-induced interaction between the partner protein incorporated inthe third fusion protein and the light-induced heterodimerized protein.The third fusion protein may comprise at least two repeated the partnerproteins. Alternately a single copy of the partner protein may be usedfor the nano-cluster formation when the partner protein can behomodimerized like PHR. The interaction between the bait protein and thetarget protein may be analyzed by determining whether the fourth fusionprotein containing the target protein the third fluorescent proteininteracts with the nanoparticles, wherein the fourth fusion protein isexpressed independent of the first to third fusion protein. In thiscase, nano-clusters are generated all the time by light irradiation andif there is an interaction between the bait protein and the targetprotein, the fluorescent pattern generated by the fourth fusion proteinwill be same with that generated by the nano-cluster formation. On thecontrary, if there is no interaction between the bait protein and thetarget protein the fluorescent pattern generated by the fourth fusionprotein will be a dispersed one unlike that generated by thenano-cluster formation.

In another aspect of the present invention, a kit for analyzinginteraction between proteins is provided, wherein the kit comprises: afirst expression vector including a first gene construct containing apromoter, a first polynucleotide encoding a first fusion proteincomprising a light-induced heterodimerized protein, a first fluorescentprotein and a self-assembled protein, wherein the first polynucleotideis operably linked to the promoter; a second expression vector includinga second gene construct containing a promoter, and a secondpolynucleotide encoding a second fusion protein comprising a partnerprotein interacting with the light-induced heterodimerized protein andforming a homodimer by itself, a second fluorescent protein and a baitprotein, wherein the second polynucleotide is operably linked to thepromoter; and a third expression vector including a third gene constructcontaining a promoter and a third polynucleotide encoding a third fusionprotein comprising a target protein and a third fluorescent protein,wherein the third polynucleotide is operably linked to the promoter;with the proviso that all the fluorescent proteins emit differentwavelengths of light and the self-assembled protein may be omitted fromthe first fusion protein when the first fluorescent protein is DsRed(FIG. 7).

In another aspect of the present invention, a kit for analyzinginteraction between proteins is provided, wherein the kit comprises: afirst expression vector including a first gene construct containing apromoter, a first polynucleotide encoding a first fusion proteincomprising a light-induced heterodimerized protein, a first fluorescentprotein and a self-assembled protein, wherein the first polynucleotideis operably linked to the promoter; a second expression vector includinga second gene construct containing a promoter, and a secondpolynucleotide encoding a second fusion protein comprising a partnerprotein interacting with the light-induced heterodimerized protein andforming a homodimer by itself, and a second fluorescent protein and amulticloning site in which a polynucleotide encoding a bait protein isoperably linked to the second polynucleotide, wherein the secondpolynucleotide is operably linked to the promoter; and a thirdexpression vector including a third gene construct containing a promoterand a third polynucleotide encoding a third fusion protein comprising athird fluorescent protein, and a multicloning site in which apolynucleotide encoding a target protein is operably linked to the thirdpolynucleotide, wherein the third polynucleotide is operably linked tothe promoter; with the proviso that all the fluorescent proteins emitdifferent wavelengths of light and the self-assembled protein may beomitted from the first fusion protein or the second fusion protein whenthe first fluorescent protein is DsRed.

According to the kit, the light-induced heterodimerized protein may beCIB, or CIBN.

According to the kit, the partner protein is a protein capable offorming a heterodimer with the light-induced protein by lightirradiation and forming a homodimer regardless of light irradiation andmay be CRY or PHR.

According to the kit, the first to third fluorescent proteins may begreen fluorescent protein (GFP), yellow fluorescent protein (YFP), redfluorescent protein (RFP), orange fluorescent protein, cyan fluorescentprotein (CFP), blue fluorescent protein (BFP), TagCFP, DsRed ortetracysteine fluorescent motif, independently.

According to the kit, the self-assembled may be ferritin, virus capsidprotein, ferritin-like protein, calcium/calmodulin-dependent proteinkinase II alpha subunit (CaMKIIα) or DsRed and the virus capsid proteinmay be a capsid protein derived from CCMV (cowpea chlorotic mottlevirus), Norwalk virus, SV40, or HPV (human papilloma virus).

According to the kit, the promoter may be a eukaryotic promoter.

FIG. 7 is a schematic overview representing the principles of a methodfor analyzing the interaction between the proteins using the nanoclusterformation according to still another embodiment of the presentinvention. As shown in FIG. 7, according to the example, a singlenanoparticle is used. The nanoparticle is generated by self-assembly ofthe first fusion protein containing forms a hetero-assemblednanoparticle between the first fusion protein comprising a light-inducedheterodimerized protein, the first fluorescent protein and theself-assembled protein, and the second fusion protein containing thepartner protein, the second fluorescent protein and the bait protein andthe self-assemble protein. In this case, the first fluorescent proteinis incorporated to either the first fusion protein or the second fusionprotein in order to determine whether the fusion proteins are expressedand nano-clusters are generated. The formation of nano-clusters isaccomplished by the light-induced interaction between the partnerprotein incorporated in the third fusion protein and the light-inducedheterodimerized protein. The third fusion protein may comprise at leasttwo repeated the partner proteins. Alternately a single copy of thepartner protein may be used for the nano-cluster formation when thepartner protein can be homodimerized like PHR. The interaction betweenthe bait protein and the target protein may be analyzed by determiningwhether the fourth fusion protein containing the target protein thethird fluorescent protein interacts with the nanoparticles, wherein thefourth fusion protein is expressed independent of the first to thirdfusion protein. In this case, nano-clusters are generated all the timeby light irradiation and if there is an interaction between the baitprotein and the target protein, the fluorescent pattern generated by thefourth fusion protein will be same with that generated by thenano-cluster formation. On the contrary, if there is no interactionbetween the bait protein and the target protein, the fluorescent patterngenerated by the fourth fusion protein will be a dispersed one unlikethat generated by the nano-cluster formation.

In another aspect of the present invention, a kit for analyzinginteraction between proteins is provided, wherein the kit comprises: afirst expression vector including a first gene construct containing apromoter and a first polynucleotide encoding a first fusion proteincomprising a self-assembled protein, a first fluorescent protein and alight-induced heterodimerized protein, wherein the first polynucleotideis operably linked to the promoter; a second expression vector includinga second gene construct containing a promoter, and a secondpolynucleotide encoding a second fusion protein comprising a partnerprotein interacting with the light-induced heterodimerized protein, asecond fluorescent protein and a bait protein, wherein the secondpolynucleotide is operably linked to the promoter; and a thirdexpression vector including a third gene construct containing a promoterand a third polynucleotide encoding a third fusion protein comprising atarget protein which is a subject to be examined whether it interactswith the bait protein or not, and a third fluorescent protein, whereinthe third polynucleotide is operably linked to the promoter;

with the proviso that all the fluorescent proteins emit differentwavelengths of light and the first fluorescent protein may be omittedfrom the first fusion protein when the self-assembled protein is DsRed(FIG. 8).

In another aspect of the present invention, a kit for analyzinginteraction between proteins is provided, wherein the kit comprises: afirst expression vector including a first gene construct containing apromoter, a first polynucleotide encoding a first fusion proteincomprising a self-assembled protein, a first fluorescent protein and alight-induced heterodimerized protein, wherein the first polynucleotideis operably linked to the promoter; a second expression vector includinga second gene construct containing a promoter, a second polynucleotideencoding a second fusion protein comprising a partner proteininteracting with the light-induced heterodimerized protein and a secondfluorescent protein, and a multicloning site in which a polynucleotideencoding a bait protein is operably linked to the second polynucleotide,wherein the second polynucleotide is operably linked to the promoter;and a third expression vector including a third gene constructcontaining a promoter, a third polynucleotide encoding a third fusionprotein comprising a third fluorescent protein, and a multicloning sitein which a polynucleotide encoding a target protein which is a subjectto be examined whether it interacts with the bait protein or not isoperably linked to the third polynucleotide, wherein the thirdpolynucleotide is operably linked to the promoter, with the proviso thatall the fluorescent proteins emit different wavelengths of light and thefirst fluorescent protein may be omitted from the first fusion proteinor the first fusion protein when the self-assembled protein is DsRed.

According to the kit, the first to third fluorescent proteins may begreen fluorescent protein (GFP), yellow fluorescent protein (YFP), redfluorescent protein (RFP), orange fluorescent protein, cyan fluorescentprotein (CFP), blue fluorescent protein (BFP), TagCFP, DsRed ortetracysteine fluorescent motif, independently.

According to the kit, the light-induced heterodimerized protein may beCIB, or CIBN.

According to the kit, the partner protein is a protein capable offorming a heterodimer with the light-induced protein by lightirradiation and forming a homodimer regardless of light irradiation andmay be CRY or PHR.

According to the kit, the self-assembled may be ferritin, virus capsidprotein, ferritin-like protein, calcium/calmodulin-dependent proteinkinase II alpha subunit (CaMKIIα) or DsRed and the virus capsid proteinmay be a capsid protein derived from CCMV (cowpea chlorotic mottlevirus), Norwalk virus, SV40, or HPV (human papilloma virus).

According to the kit, the promoter may be a eukaryotic promoter.

As shown in FIG. 8, the present invention may be implemented by using asingle nanoparticle comprising the light-induced heterodimerized proteincapable of forming a homodimer regardless of light irradiation as wellas forming a heterodimer with the partner protein by light irradiationand the self-assemble protein. Particularly, the embodiment of thepresent invention may be implemented by constructing the firstexpression vector capable of expressing the first fusion proteinconsisting of the self-assembled protein, the first fluorescent proteinand the light-induced heterodimerized protein; the second expressionvector capable of expressing the second fusion protein consisting of thepartner protein interacting with the light-induced heterodimerizedprotein, the second fluorescent protein and a bait protein, and thethird expression vector capable of expressing the third fusion proteinconsisting of the target protein to be examined whether it interactswith the bait protein or not and the third fluorescent protein. Thelight-induced heterodimerized protein capable of forming homodimerregardless of light irradiation may be CRY or PHR, an N-terminalfragment of the CRY, and the partner protein may be CIB or CIBN, anN-terminal fragment of CIB. If cells are cotransfected with theexpression vectors and irradiating light having wavelengths capable ofinducing heterodimerizing of the light-induced heterodimerized proteinand the partner protein, light-induced nano-clusters are generated andin this case the fluorescent patterns by the first fluorescent proteinand the second fluorescent protein are same with each other. In otherwords, the fluorescence emitted by the first fluorescent protein isdisplayed in a pattern of strong spots in the position where thecorresponding fusion proteins are expressed according to thenano-cluster formation. In the meantime, the fluorescence emitted by thethird fluorescent protein linked to the target protein is displayeddifferently depending on the interaction between the bait protein andthe target protein. In other words, if the target protein interacts withthe bait protein, the fluorescence emitted by the third fluorescentprotein is displayed in the same pattern that emitted by the secondfluorescent protein and if there is no interaction between the targetprotein and the bait protein, the fluorescent protein emitted by thethird fluorescent protein will be displayed in a dispersed pattern inthe position where the third fusion proteins are expressed after lightirradiation.

In addition, the method of analyzing protein-protein interaction may beused for screening a substance regulating interprotein interaction, i.e.a substance promoting or inhibiting certain interprotein interaction.Life phenomena are represented by the result of an interproteininteraction, i.g. an interaction between a receptor and a ligand bindingthereto and an abnormal interaction between these proteins may result inpathological condition. Therefore, the kit and method of the presentinvention may be used as a kit and method for screening of therapeuticcandidates in cellular level efficiently.

Indeed, the present inventors co-expressed a fusion protein in whichCIBN is fused to an activation domain (AD) of CalMKIIα, a fusion proteinin which PHR is fused to Hras, and RBD_(raf1) in cells and irradiatedblue light having 488 nm which induces the interaction between CIBN andPHR. As a result, the interaction between Hras and RBD_(raf1) wasconfirmed by analyzing of fluorescence pattern shown in the cells (SeeFIG. 18).

In another aspect to the present invention, a method for inhibiting atarget protein reversibly using light-induced protein nano-clusterformation is provided, wherein the method comprises: co-expressing afusion protein comprising a self-assembled protein and a light-inducedheterodimerized protein, a partner protein capable of heterodimerizingwith light-induced heterodimerized protein, and a bait proteininteracting with a target protein in a cell or a subject expressing thetarget protein as an inherent protein; and inducing a proteinnano-cluster formation by irradiating light having wavelength capable ofinducing heterodimerizing between the light-induced heterodimerizedprotein and the partner protein to the cell or the subject

According to the method, the self-assembled protein may be ferritin,virus capsid protein, ferritin-like protein,calcium/calmodulin-dependent protein kinase II alpha subunit (CaMKIIα)or DsRed and the virus capsid protein may be a capsid protein derivedfrom CCMV (cowpea chlorotic mottle virus), Norwalk virus, SV40, or HPV(human papilloma virus).

According to the method, at least one protein among the fusion protein,the partner protein and the bait protein may further comprise afluorescent protein in order to determine whether the nano-cluster isgenerated or not. In this case, the fluorescent protein may be greenfluorescent protein (GFP), yellow fluorescent protein (YFP), redfluorescent protein (RFP), orange fluorescent protein, cyan fluorescentprotein (CFP), blue fluorescent protein (BFP), or tetracysteinefluorescent motif. The green fluorescent protein may be EGFP, Emerald,Superfolder GFP, Azami Green, TagGFP, TurboGFP, ZsGreen or T-Sapphire.The yellow fluorescent protein may be EYFP, Topaz, Venus, mCitrine,Ypet, TagYFP, PhiYFP, mBanana, or ZsYellow1. The red fluorescent proteinmay be mRuby, mApple, mStrawberry, AsRed2 or mRFP. The orangefluorescent protein may be Kusabira Orange, Kusabira Orange2, mOrange,mOrange2, dTomato, dTomato-Tandem, TagRFP, TagRFP-T, DsRed, DsRed2,DsRed-Express, DsRed-Monomer or mTangerine. The cyan fluorescent proteinmay be ECFP, mECFP, mCerulean, CyPet, AmCyan1, Midori-Ishi Cyan, TagCFP,or mTFP1. The blue fluorescent protein may be EBFP, EBFP2, Azurite ormTagBFP. The far red fluorescent protein may be mPlum, mCherry, thedKeima-Tandem, JRed, mRaspberry, HcRed1, HcRed-Tandem, or AQ143. Thetetracysteine fluorescent motif may be a polypeptide including an aminoacid sequence of Cys-Cys-Xaa-Xaa-Xaa-Cys-Cys (SEQ ID NO: 1), wherein theXaa is one of any amino acids except cysteine.

According to the method, the light-induced heterodimerized protein maybe CIB, CIBN, PhyB, PIF, FKF1, GIGANTEA, CRY, or PHR.

According to the method, the partner protein may be CRY or PHR when thelight-induced heterodimerized protein is CIB or CIBN, the partnerprotein may be PIF when the light-induced heterodimerized protein isPhyB, the partner protein may be GIFANTEA when the light-inducedheterodimerized protein is FKF1, the partner protein may be CIB, CIBN,PhyB, PIF, FKF1, GIGANTEA, CRY, or PHR, and the partner protein may beCRY or PHR when the light-induced heterodimerized protein is CIB orCIBN, the partner protein may be PIF when the light-inducedheterodimerized protein is PhyB, the partner protein may be GIFANTEAwhen the light-induced heterodimerized protein is FKF1, the partnerprotein may be CIB or CIBN when the light-induced heterodimerizedprotein is CRY or PHR, the partner protein may be PIF when thelight-induced heterodimerized protein is PhyB, or the partner proteinmay be FKF1 when the light-induced heterodimerized protein is GIGANTEA.The PIF may be PIF3 or PIF6.

According to the method, the light-induced heterodimerized protein orthe partner protein may a protein capable of forming a heterodimer withthe light-induced protein by light irradiation and forming a homodimerregardless of light irradiation. In this case the light-inducedheterodimerized protein or the partner protein may be CRY or PHR.

In another aspect of the present invention, a method for inhibiting atarget protein reversibly using light-induced protein nano-clusterformation is provided, wherein the method comprises: co-expressing afirst fusion protein comprising a first self-assembled protein and alight-induced heterodimerized protein, a second fusion proteincomprising a second self-assembled protein and a partner protein capableof heterodimerizing with the light-induced heterodimerized protein, anda bait protein interacting with a target protein in a cell or a subjectexpressing the target protein as an inherent protein; and inducing aprotein nano-cluster formation by irradiating light having wavelengthcapable of inducing heterodimerizing between the light-inducedheterodimerized protein and the partner protein to the cell or thesubject, wherein the bait protein is expressed as a fusion protein withthe first self-assembled protein or the second self-assembled protein.

According to the method, the first self-assembled protein and the secondself-assembled protein may be independently ferritin, virus capsidprotein, ferritin-like protein, calcium/calmodulin-dependent proteinkinase II alpha subunit (CaMKIIα) or DsRed and the virus capsid proteinmay be a capsid protein derived from CCMV (cowpea chlorotic mottlevirus), Norwalk virus, SV40, or HPV (human papilloma virus). In thiscase, when the first self-assembled protein and the secondself-assembled protein are used at the same time, the firstself-assembled protein and the second self-assembled protein may be sameor different

According to the method, at least one protein among the fusion protein,the partner protein and the bait protein may further comprise afluorescent protein in order to determine whether the nano-cluster isgenerated or not. In this case, the fluorescent protein may be greenfluorescent protein (GFP), yellow fluorescent protein (YFP), redfluorescent protein (RFP), orange fluorescent protein, cyan fluorescentprotein (CFP), blue fluorescent protein (BFP), or tetracysteinefluorescent motif. The green fluorescent protein may be EGFP, Emerald,Superfolder GFP, Azami Green, TagGFP, TurboGFP, ZsGreen or T-Sapphire.The yellow fluorescent protein may be EYFP, Topaz, Venus, mCitrine,Ypet, TagYFP, PhiYFP, mBanana, or ZsYellow1. The red fluorescent proteinmay be mRuby, mApple, mStrawberry, AsRed2 or mRFP. The orangefluorescent protein may be Kusabira Orange, Kusabira Orange2, mOrange,mOrange2, dTomato, dTomato-Tandem, TagRFP, TagRFP-T, DsRed, DsRed2,DsRed-Express, DsRed-Monomer or mTangerine. The cyan fluorescent proteinmay be ECFP, mECFP, mCerulean, CyPet, AmCyan1, Midori-Ishi Cyan, TagCFP,or mTFP1. The blue fluorescent protein may be EBFP, EBFP2, Azurite ormTagBFP. The far red fluorescent protein may be mPlum, mCherry, thedKeima-Tandem, JRed, mRaspberry, HcRed1, HcRed-Tandem, or AQ143. Thetetracysteine fluorescent motif may be a polypeptide including an aminoacid sequence of Cys-Cys-Xaa-Xaa-Xaa-Cys-Cys (SEQ ID NO: 1), wherein theXaa is one of any amino acids except cysteine.

According to the method, the light-induced heterodimerized protein maybe CIB, CIBN, PhyB, PIF, FKF1, GIGANTEA, CRY, or PHR.

According to the method, the partner protein may be CRY or PHR when thelight-induced heterodimerized protein is CIB or CIBN, the partnerprotein may be PIF when the light-induced heterodimerized protein isPhyB, the partner protein may be GIFANTEA when the light-inducedheterodimerized protein is FKF1, the partner protein may be CIB, CIBN,PhyB, PIF, FKF1, GIGANTEA, CRY, or PHR, and the partner protein may beCRY or PHR when the light-induced heterodimerized protein is CIB orCIBN, the partner protein may be PIF when the light-inducedheterodimerized protein is PhyB, the partner protein may be GIFANTEAwhen the light-induced heterodimerized protein is FKF1, the partnerprotein may be CIB or CIBN when the light-induced heterodimerizedprotein is CRY or PHR, the partner protein may be PIF when thelight-induced heterodimerized protein is PhyB, or the partner proteinmay be FKF1 when the light-induced heterodimerized protein is GIGANTEA.The PIF may be PIF3 or PIF6.

According to the method, the light-induced heterodimerized protein orthe partner protein may form a homodimer regardless of light irradiationby itself. In this case the light-induced heterodimerized protein or thepartner protein may be CRY or PHR.

In another aspect of the present invention, a kit for inhibiting atarget protein reversibly using light-induced protein nano-clusterformation is provided, wherein the kit comprises: a first expressionvector including a first gene construct containing a promoter and afirst polynucleotide encoding a fusion protein comprising aself-assembled protein and a light-induced heterodimerized protein,wherein the first polynucleotide is operably linked to the promoter; asecond expression vector including a second gene construct containing apromoter, and a second polynucleotide encoding a partner protein capableof heterodimerizing with light-induced heterodimerized protein, whereinthe second polynucleotide is operably linked to the promoter; and athird expression vector including a third gene construct containing apromoter and a third polynucleotide encoding a bait protein interactingwith the target protein, wherein the third polynucleotide is operablylinked to the promoter, wherein the bait protein is expressed as afusion protein with the self-assembled protein or the partner protein.

According to the kit, the self-assembled protein may be ferritin, viruscapsid protein, ferritin-like protein, calcium/calmodulin-dependentprotein kinase II alpha subunit (CaMKIIα) or DsRed and the virus capsidprotein may be a capsid protein derived from CCMV (cowpea chloroticmottle virus), Norwalk virus, SV40, or HPV (human papilloma virus).

According to the kit, at least one protein among the fusion protein, thepartner protein and the bait protein may further comprise a fluorescentprotein in order to determine whether the nano-cluster is generated ornot. In this case, the fluorescent protein may be green fluorescentprotein (GFP), yellow fluorescent protein (YFP), red fluorescent protein(RFP), orange fluorescent protein, cyan fluorescent protein (CFP), bluefluorescent protein (BFP), or tetracysteine fluorescent motif. The greenfluorescent protein may be EGFP, Emerald, Superfolder GFP, Azami Green,TagGFP, TurboGFP, ZsGreen or T-Sapphire. The yellow fluorescent proteinmay be EYFP, Topaz, Venus, mCitrine, Ypet, TagYFP, PhiYFP, mBanana, orZsYellow1. The red fluorescent protein may be mRuby, mApple,mStrawberry, AsRed2 or mRFP. The orange fluorescent protein may beKusabira Orange, Kusabira Orange2, mOrange, mOrange2, dTomato,dTomato-Tandem, TagRFP, TagRFP-T, DsRed, DsRed2, DsRed-Express,DsRed-Monomer or mTangerine. The cyan fluorescent protein may be ECFP,mECFP mCerulean, CyPet, AmCyan1, Midori-Ishi Cyan, TagCFP, or mTFP1. Theblue fluorescent protein may be EBFP, EBFP2, Azurite or mTagBFP. The farred fluorescent protein may be mPlum, mCherry, the dKeima-Tandem, JRed,mRaspberry, HcRed1, HcRed-Tandem, or AQ143. The tetracysteinefluorescent motif may be a polypeptide including an amino acid sequenceof Cys-Cys-Xaa-Xaa-Xaa-Cys-Cys (SEQ ID NO: 1), wherein the Xaa is one ofany amino acids except cysteine.

According to the kit, the light-induced heterodimerized protein may beCIB, CIBN, PhyB, PIF, FKF1, GIGANTEA, CRY, or PHR.

According to the kit, the partner protein may be CRY or PHR when thelight-induced heterodimerized protein is CIB or CIBN, the partnerprotein may be PIF when the light-induced heterodimerized protein isPhyB, the partner protein may be GIFANTEA when the light-inducedheterodimerized protein is FKF1, the partner protein may be CIB, CIBN,PhyB, PIF, FKF1, GIGANTEA, CRY, or PHR, and the partner protein may beCRY or PHR when the light-induced heterodimerized protein is CIB orCIBN, the partner protein may be PIF when the light-inducedheterodimerized protein is PhyB, the partner protein may be GIFANTEAwhen the light-induced heterodimerized protein is FKF1, the partnerprotein may be CIB or CIBN when the light-induced heterodimerizedprotein is CRY or PHR, the partner protein may be PIF when thelight-induced heterodimerized protein is PhyB, or the partner proteinmay be FKF1 when the light-induced heterodimerized protein is GIGANTEA.The PIF may be PIF3 or PIF6.

According to the kit, the light-induced heterodimerized protein or thepartner protein may form a homodimer regardless of light irradiation byitself. In this case the light-induced heterodimerized protein or thepartner protein may be CRY or PHR.

In another aspect of the present invention, a kit for inhibiting atarget protein reversibly using light-induced protein nano-clusterformation is provided, wherein the kit comprises: a first expressionvector including a first gene construct containing a promoter and afirst polynucleotide encoding a first fusion protein comprising a firstself-assembled protein and a light-induced heterodimerized protein,wherein the first polynucleotide is operably linked to the promoter; asecond expression vector including a second gene construct containing apromoter and a second polynucleotide encoding a second fusion proteincomprising a second self-assembled protein and a partner protein capableof heterodimerizing with the light-induced heterodimerized protein,wherein the second polynucleotide is operably linked to the promoter;and a third expression vector including a third gene constructcontaining a promoter and a multicloning site for inserting a thirdpolynucleotide encoding a bait protein interacting with the targetprotein, wherein the third polynucleotide is operably linked to thepromoter, and wherein the bait protein is expressed as a fusion proteinwith the self-assembled protein or the partner protein.

According to the kit, the first self-assembled protein and the secondself-assembled protein may be independently ferritin, virus capsidprotein, ferritin-like protein, calcium/calmodulin-dependent proteinkinase II alpha subunit (CaMKIIα) or DsRed and the virus capsid proteinmay be a capsid protein derived from CCMV (cowpea chlorotic mottlevirus), Norwalk virus, SV40, or HPV (human papilloma virus).

According to the kit, at least one protein among the fusion protein, thepartner protein and the bait protein may further comprise a fluorescentprotein in order to determine whether the nano-cluster is generated ornot. In this case, the fluorescent protein may be green fluorescentprotein (GFP), yellow fluorescent protein (YFP), red fluorescent protein(RFP), orange fluorescent protein, cyan fluorescent protein (CFP), bluefluorescent protein (BFP), or tetracysteine fluorescent motif. The greenfluorescent protein may be EGFP, Emerald, Superfolder GFP, Azami Green,TagGFP, TurboGFP, ZsGreen or T-Sapphire. The yellow fluorescent proteinmay be EYFP, Topaz, Venus, mCitrine, Ypet, TagYFP, PhiYFP, mBanana, orZsYellow1. The red fluorescent protein may be mRuby, mApple,mStrawberry, AsRed2 or mRFP. The orange fluorescent protein may beKusabira Orange, Kusabira Orange2, mOrange, mOrange2, dTomato,dTomato-Tandem, TagRFP, TagRFP-T, DsRed, DsRed2, DsRed-Express,DsRed-Monomer or mTangerine. The cyan fluorescent protein may be ECFP,mECFP, mCerulean, CyPet, AmCyan1, Midori-Ishi Cyan, TagCFP, or mTFP1.The blue fluorescent protein may be EBFP, EBFP2, Azurite or mTagBFP. Thefar red fluorescent protein may be mPlum, mCherry, the dKeima-Tandem,JRed, mRaspberry, HcRed1, HcRed-Tandem, or AQ143. The tetracysteinefluorescent motif may be a polypeptide including an amino acid sequenceof Cys-Cys-Xaa-Xaa-Xaa-Cys-Cys (SEQ ID NO: 1), wherein the Xaa is one ofany amino acids except cysteine.

According to the kit, the light-induced heterodimerized protein may beCIB, CIBN, PhyB, PIF, FKF1, GIGANTEA, CRY, or PHR.

According to the kit, the partner protein may be CRY or PHR when thelight-induced heterodimerized protein is CIB or CIBN, the partnerprotein may be PIF when the light-induced heterodimerized protein isPhyB, the partner protein may be GIFANTEA when the light-inducedheterodimerized protein is FKF1, the partner protein may be CIB, CIBN,PhyB, PIF, FKF1, GIGANTEA, CRY, or PHR, and the partner protein may beCRY or PHR when the light-induced heterodimerized protein is CIB orCIBN, the partner protein may be PIF when the light-inducedheterodimerized protein is PhyB, the partner protein may be GIFANTEAwhen the light-induced heterodimerized protein is FKF1, the partnerprotein may be CIB or CIBN when the light-induced heterodimerizedprotein is CRY or PHR, the partner protein may be PIF when thelight-induced heterodimerized protein is PhyB, or the partner proteinmay be FKF1 when the light-induced heterodimerized protein is GIGANTEA.The PIF may be PIF3 or PIF6.

According to the kit, the light-induced heterodimerized protein or thepartner protein may form a homodimer regardless of light irradiation byitself. In this case the light-induced heterodimerized protein or thepartner protein may be CRY or PHR.

In another aspect of the present invention, a kit for inhibiting atarget protein reversibly using light-induced protein nano-clusterformation is provided, wherein the kit comprises: a first expressionvector including a first gene construct containing a promoter and afirst polynucleotide encoding a fusion protein comprising aself-assembled protein and a light-induced heterodimerized protein,wherein the first polynucleotide is operably linked to the promoter; asecond expression vector including a second gene construct containing apromoter and a second polynucleotide encoding a partner protein capableof heterodimerizing with the light-induced heterodimerized protein,wherein the second polynucleotide is operably linked to the promoter;and a third expression vector including a third gene constructcontaining a promoter and a multicloning site for inserting a thirdpolynucleotide encoding a bait protein interacting with the targetprotein, wherein the third polynucleotide is operably linked to thepromoter, wherein the bait protein is expressed as a fusion protein withthe self-assembled protein or the partner protein.

According to the kit, the self-assembled protein may be ferritin, viruscapsid protein, ferritin-like protein, calcium/calmodulin-dependentprotein kinase II alpha subunit (CaMKIIα) or DsRed and the virus capsidprotein may be a capsid protein derived from CCMV (cowpea chloroticmottle virus), Norwalk virus, SV40, or HPV (human papilloma virus).

According to the kit, at least one protein among the fusion protein, thepartner protein and the bait protein may further comprise a fluorescentprotein in order to determine whether the nano-cluster is generated ornot. In this case, the fluorescent protein may be green fluorescentprotein (GFP), yellow fluorescent protein (YFP), red fluorescent protein(RFP), orange fluorescent protein, cyan fluorescent protein (CFP), bluefluorescent protein (BFP), or tetracysteine fluorescent motif. The greenfluorescent protein may be EGFP, Emerald, Superfolder GFP, Azami Green,TagGFP, TurboGFP, ZsGreen or T-Sapphire. The yellow fluorescent proteinmay be EYFP, Topaz, Venus, mCitrine, Ypet, TagYFP, PhiYFP, mBanana, orZsYellow1. The red fluorescent protein may be mRuby, mApple,mStrawberry, AsRed2 or mRFP. The orange fluorescent protein may beKusabira Orange, Kusabira Orange2, mOrange, mOrange2, dTomato,dTomato-Tandem, TagRFP, TagRFP-T, DsRed, DsRed2, DsRed-Express,DsRed-Monomer or mTangerine. The cyan fluorescent protein may be ECFP,mECFP, mCerulean, CyPet, AmCyan1, Midori-Ishi Cyan, TagCFP, or mTFP1.The blue fluorescent protein may be EBFP, EBFP2, Azurite or mTagBFP. Thefar red fluorescent protein may be mPlum, mCherry, the dKeima-Tandem,JRed, mRaspberry, HcRed1, HcRed-Tandem, or AQ143. The tetracysteinefluorescent motif may be a polypeptide including an amino acid sequenceof Cys-Cys-Xaa-Xaa-Xaa-Cys-Cys (SEQ ID NO: 1), wherein the Xaa is one ofany amino acids except cysteine.

According to the kit, the light-induced heterodimerized protein may beCIB, CIBN, PhyB, PIF, FKF1, GIGANTEA, CRY, or PHR.

According to the kit, the partner protein may be CRY or PHR when thelight-induced heterodimerized protein is CIB or CIBN, the partnerprotein may be PIF when the light-induced heterodimerized protein isPhyB, the partner protein may be GIFANTEA when the light-inducedheterodimerized protein is FKF1, the partner protein may be CIB, CIBN,PhyB, PIF, FKF1, GIGANTEA, CRY, or PHR, and the partner protein may beCRY or PHR when the light-induced heterodimerized protein is CIB orCIBN, the partner protein may be PIF when the light-inducedheterodimerized protein is PhyB, the partner protein may be GIFANTEAwhen the light-induced heterodimerized protein is FKF1, the partnerprotein may be CIB or CIBN when the light-induced heterodimerizedprotein is CRY or PHR, the partner protein may be PIF when thelight-induced heterodimerized protein is PhyB, or the partner proteinmay be FKF1 when the light-induced heterodimerized protein is GIGANTEA.The PIF may be PIF3 or PIF6.

According to the kit, the light-induced heterodimerized protein or thepartner protein may form a homodimer regardless of light irradiation byitself. In this case the light-induced heterodimerized protein or thepartner protein may be CRY or PHR.

In another aspect of the present invention, a kit for inhibiting atarget protein reversibly using light-induced protein nano-clusterformation is provided, wherein the kit comprises: a first expressionvector including a first gene construct containing a promoter and afirst polynucleotide encoding a first fusion protein comprising a firstself-assembled protein and a light-induced heterodimerized protein,wherein the first polynucleotide is operably linked to the promoter; asecond expression vector including a second gene construct containing apromoter and a second polynucleotide encoding a second fusion proteincomprising a second self-assembled protein and a partner protein capableof heterodimerizing with the light-induced heterodimerized protein,wherein the second polynucleotide is operably linked to the promoter;and a third expression vector including a third gene constructcomprising a promoter and a third polynucleotide encoding a bait proteininteracting with the target protein, wherein the third polynucleotide isoperably linked to the promoter, wherein the bait protein is expressedas a fusion protein with the first self-assembled protein or the secondself-assembled protein.

According to the kit, the first self-assembled protein and the secondself-assembled protein may be independently ferritin, virus capsidprotein, ferritin-like protein, calcium/calmodulin-dependent proteinkinase II alpha subunit (CaMKIIα) or DsRed and the virus capsid proteinmay be a capsid protein derived from CCMV (cowpea chlorotic mottlevirus), Norwalk virus, SV40, or HPV (human papilloma virus).

According to the kit, at least one protein among the fusion protein, thepartner protein and the bait protein may further comprise a fluorescentprotein in order to determine whether the nano-cluster is generated ornot. In this case, the fluorescent protein may be green fluorescentprotein (GFP), yellow fluorescent protein (YFP), red fluorescent protein(RFP), orange fluorescent protein, cyan fluorescent protein (CFP), bluefluorescent protein (BFP), or tetracysteine fluorescent motif. The greenfluorescent protein may be EGFP, Emerald, Superfolder GFP, Azami Green,TagGFP, TurboGFP, ZsGreen or T-Sapphire. The yellow fluorescent proteinmay be EYFP, Topaz, Venus, mCitrine, Ypet, TagYFP, PhiYFP, mBanana, orZsYellow1. The red fluorescent protein may be mRuby, mApple,mStrawberry, AsRed2 or mRFP. The orange fluorescent protein may beKusabira Orange, Kusabira Orange2, mOrange, mOrange2, dTomato,dTomato-Tandem, TagRFP, TagRFP-T, DsRed, DsRed2, DsRed-Express,DsRed-Monomer or mTangenne. The cyan fluorescent protein may be ECFP,mECFP, mCerulean, CyPet, AmCyan1, Midori-Ishi Cyan, TagCFP, or mTFP1.The blue fluorescent protein may be EBFP, EBFP2, Azurite or mTagBFP. Thefar red fluorescent protein may be mPlum, mCherry, the dKeima-Tandem,JRed, mRaspberry, HcRed1, HcRed-Tandem, or AQ143. The tetracysteinefluorescent motif may be a polypeptide including an amino acid sequenceof Cys-Cys-Xaa-Xaa-Xaa-Cys-Cys (SEQ ID NO: 1), wherein the Xaa is one ofany amino acids except cysteine.

According to the kit, the light-induced heterodimerized protein may beCIB, CIBN, PhyB, PIF, FKF1, GIGANTEA, CRY, or PHR.

According to the kit, the partner protein may be CRY or PHR when thelight-induced heterodimerized protein is CIB or CIBN, the partnerprotein may be PIF when the light-induced heterodimerized protein isPhyB, the partner protein may be GIFANTEA when the light-inducedheterodimerized protein is FKF1, the partner protein may be CIB, CIBN,PhyB, PIF, FKF1, GIGANTEA, CRY, or PHR, and the partner protein may beCRY or PHR when the light-induced heterodimerized protein is CIB orCIBN, the partner protein may be PIF when the light-inducedheterodimerized protein is PhyB, the partner protein may be GIFANTEAwhen the light-induced heterodimerized protein is FKF1, the partnerprotein may be CIB or CIBN when the light-induced heterodimerizedprotein is CRY or PHR, the partner protein may be PIF when thelight-induced heterodimerized protein is PhyBC or the partner proteinmay be FKF1 when the light-induced heterodimerized protein is GIGANTEA.The PIF may be PIF3 or PIF6.

According to the kit, the light-induced heterodimerized protein or thepartner protein may form a homodimer regardless of light irradiation byitself. In this case the light-induced heterodimerized protein or thepartner protein may be CRY or PHR.

In an aspect of the present invention, a method for inhibiting a targetprotein reversibly using light-induced nanocluster formation, whereinthe method comprises: co-expressing a first fusion protein includingself-assembled protein and a light-induced heterodimerzable protein, apartner protein forming a heterodimer with the light-inducedheterodimerzable protein, and a target protein in a cell or a subject;and inducing light-induced formation of nanocluster by irradiating lighthaving wavelength capable of forming the heterodimer between thelight-induced heterodimerized protein and the partner protein, whereinthe target protein is expressed as a fusion protein with theself-assembled protein or the partner protein.

According to the method, the first self-assembled protein and the secondself-assembled protein may be independently ferritin, virus capsidprotein, ferritin-like protein, calcium/calmodulin-dependent proteinkinase II alpha subunit (CaMKIIα) or DsRed and the virus capsid proteinmay be a capsid protein derived from CCMV (cowpea chlorotic mottlevirus), Norwalk virus, SV40, or HPV (human papilloma virus).

According to the method, at least one protein among the fusion protein,the partner protein and the bait protein may further comprise afluorescent protein in order to determine whether the nano-cluster isgenerated or not. In this case, the fluorescent protein may be greenfluorescent protein (GFP), yellow fluorescent protein (YFP), redfluorescent protein (RFP), orange fluorescent protein, cyan fluorescentprotein (CFP), blue fluorescent protein (BFP), or tetracysteinefluorescent motif. The green fluorescent protein may be EGFP, Emerald,Superfolder GFP, Azami Green, TagGFP, TurboGFP, ZsGreen or T-Sapphire.The yellow fluorescent protein may be EYFP, Topaz, Venus, mCitrine,Ypet, TagYFP, PhiYFP, mBanana, or ZsYellow1. The red fluorescent proteinmay be mRuby, mApple, mStrawberry, AsRed2 or mRFP. The orangefluorescent protein may be Kusabira Orange, Kusabira Orange2, mOrange,mOrange2, dTomato, dTomato-Tandem, TagRFP, TagRFP-T, DsRed, DsRed2,DsRed-Express, DsRed-Monomer or mTangerine. The cyan fluorescent proteinmay be ECFP, mECFP, mCerulean, CyPet. AmCyan1, Midori-Ishi Cyan, TagCFP,or mTFP1. The blue fluorescent protein may be EBFP, EBFP2, Azurite ormTagBFP. The far red fluorescent protein may be mPlum, mCherry, thedKeima-Tandem, JRed, mRaspberry, HcRed1, HcRed-Tandem, or AQ143. Thetetracysteine fluorescent motif may be a polypeptide including an aminoacid sequence of Cys-Cys-Xaa-Xaa-Xaa-Cys-Cys (SEQ ID NO: 1), wherein theXaa is one of any amino acids except cysteine.

According to the method, the light-induced heterodimerized protein maybe CIB, CIBN, PhyB, PIF, FKF1, GIGANTEA, CRY, or PHR.

According to the method, the partner protein may be CRY or PHR when thelight-induced heterodimerized protein is CIB or CIBN, the partnerprotein may be PIF when the light-induced heterodimerized protein isPhyB, the partner protein may be GIFANTEA when the light-inducedheterodimerized protein is FKF1, the partner protein may be CIB, CIBN.PhyB, PIF, FKF1, GIGANTEA, CRY, or PHR, and the partner protein may beCRY or PHR when the light-induced heterodimerized protein is CIB orCIBN, the partner protein may be PIF when the light-inducedheterodimerized protein is PhyB, the partner protein may be GIFANTEAwhen the light-induced heterodimerized protein is FKF1, the partnerprotein may be CIB or CIBN when the light-induced heterodimerizedprotein is CRY or PHR, the partner protein may be PIF when thelight-induced heterodimerized protein is PhyB, or the partner proteinmay be FKF1 when the light-induced heterodimerized protein is GIGANTEA.The PIF may be PIF3 or PIF6.

According to the method, the light-induced heterodimerized protein orthe partner protein may form a homodimer regardless of light irradiationby itself. In this case the light-induced heterodimerized protein or thepartner protein may be CRY or PHR.

In another aspect of the present invention, a kit for inhibiting atarget protein reversibly using light-induced protein nano-clusterformation is provided, wherein the kit comprises: a first expressionvector including a first gene construct containing a promoter and afirst polynucleotide encoding a fusion protein comprising aself-assembled protein and a light-induced heterodimerized protein,wherein the first polynucleotide is operably linked to the promoter; asecond expression vector including a second gene construct containing apromoter and a second polynucleotide encoding a second fusion proteincomprising a partner protein capable of heterodimerizing with thelight-induced heterodimerized protein, wherein the second polynucleotideis operably linked to the promoter; and optionally a third expressionvector including a third gene construct containing a promoter and athird polynucleotide encoding a third fusion protein containing thetarget protein and the self-assembled protein, wherein the thirdpolynucleotide is linked operably to the promoter, or wherein the targetprotein is included in the second fusion protein instead of comprisingthe third polynucleotide.

According to the kit, the self-assembled protein may be ferritin, viruscapsid protein, ferritin-like protein, calcium/calmodulin-dependentprotein kinase II alpha subunit (CaMKIIα) or DsRed and the virus capsidprotein may be a capsid protein derived from CCMV (cowpea chloroticmottle virus), Norwalk virus, SV40, or HPV (human papilloma virus).

According to the kit, at least one protein among the fusion protein, thepartner protein and the bait protein may further comprise a fluorescentprotein in order to determine whether the nano-cluster is generated ornot. In this case, the fluorescent protein may be green fluorescentprotein (GFP), yellow fluorescent protein (YFP), red fluorescent protein(RFP), orange fluorescent protein, cyan fluorescent protein (CFP), bluefluorescent protein (BFP), or tetracysteine fluorescent motif. The greenfluorescent protein may be EGFP, Emerald, Superfolder GFP, Azami Green,TagGFP, TurboGFP, ZsGreen or T-Sapphire. The yellow fluorescent proteinmay be EYFP, Topaz, Venus, mCitrine, Ypet, TagYFP, PhiYFP, mBanana, orZsYellow1. The red fluorescent protein may be mRuby, mApple,mStrawberry, AsRed2 or mRFP. The orange fluorescent protein may beKusabira Orange, Kusabira Orange2, mOrange, mOrange2, dTomato,dTomato-Tandem, TagRFP, TagRFP-T, DsRed. DsRed2, DsRed-Express,DsRed-Monomer or mTangerine. The cyan fluorescent protein may be ECFP,mECFP, mCerulean, CyPet, AmCyan1, Midori-Ishi Cyan, TagCFP, or mTFP1.The blue fluorescent protein may be EBFP, EBFP2, Azurite or mTagBFP. Thefar red fluorescent protein may be mPlum, mCherry, the dKeima-Tandem,JRed, mRaspberry, HcRed1, HcRed-Tandem, or AQ143. The tetracysteinefluorescent motif may be a polypeptide including an amino acid sequenceof Cys-Cys-Xaa-Xaa-Xaa-Cys-Cys (SEQ ID NO: 1), wherein the Xaa is one ofany amino acids except cysteine.

According to the kit, the light-induced heterodimerized protein may beCIB, CIBN, PhyB, PIF, FKF1, GIGANTEA, CRY, or PHR.

According to the kit, the partner protein may be CRY or PHR when thelight-induced heterodimerized protein is CIB or CIBN, the partnerprotein may be PIF when the light-induced heterodimerized protein isPhyB, the partner protein may be GIFANTEA when the light-inducedheterodimerized protein is FKF1, the partner protein may be CIB, CIBN,PhyB, PIF, FKF1, GIGANTEA, CRY, or PHR, and the partner protein may beCRY or PHR when the light-induced heterodimerized protein is CIB orCIBN, the partner protein may be PIF when the light-inducedheterodimerized protein is PhyB, the partner protein may be GIFANTEAwhen the light-induced heterodimerized protein is FKF1, the partnerprotein may be CIB or CIBN when the light-induced heterodimerizedprotein is CRY or PHR, the partner protein may be PIF when thelight-induced heterodimerized protein is PhyB, or the partner proteinmay be FKF1 when the light-induced heterodimerized protein is GIGANTEA.The PIF may be PIF3 or PIF6.

According to the kit, the light-induced heterodimerized protein or thepartner protein may form a homodimer regardless of light irradiation byitself. In this case the light-induced heterodimerized protein or thepartner protein may be CRY or PHR.

The methods and kits for inhibiting function of a protein reversibly arehereinafter described in more detail by accompanying drawings.

FIG. 9 is a schematic overview representing the principles of a methodfor inhibiting a target protein using the nanocluster formationaccording to an embodiment of the present invention. When nanoparticlescontaining a bait protein and a light-induced heterodimerized proteinand a partner protein forming a heterodimer with the light-inducedheterodimrized protein by light irradiation are co-expressed in a celland light having wavelength capable of inducing the formation of aheterodimer between the light-induced heterodimerized protein and thepartner protein is irradiated and nano-clusters are generated thereby,the target protein captured by the bait protein are confined in thenano-clusters.

FIG. 10 is a schematic overiew representing the principles of theinhibition of a particular protein by entrapment of the target proteininteracting with a partner protein via the formation of nanoclustersbetween the two proteins by irradiating light having a particularwavelength within partial active region of a cell. Since only aparticular region in a cell may be irradiated using laser beam, themethod of the present invention may be used for examining effect oninhibiting function of a protein in a particular region of a cellusefully.

FIG. 11 is a schematic overview representing the principles of theformation of nanoclusters within multicellular area such as a subjectand a tissue and the inhibition of a particular protein within the areaby irradiating light having a particular wavelength to area. The methodof the present invention is a very useful method with regard to beingcapable of observing comparatively changes of an experimental group anda control group after light irradiation.

FIG. 12 is a schematic diagram illustrating conceptually the formationof nano-clusters and the regulation of molecular function of aparticular protein thereby by light irradiation in accordance with anembodiment of the present invention. As shown in FIG. 12, thenanoparticle is a hetero-complex generated by self-assembly of a fusionprotein comprising a self-assembled protein (SAP) and a light-inducedheterodimerized protein (e.g., CIBN), and a fusion protein comprisingthe self-assembled protein and a bait protein and the bait protein takesa role as a bait for confining a target protein expressed inherently ina cell and the light-induced heterodimerized proteins form nano-clustersby interacting with partner proteins forming homodimers (e.g. PHR) andthe target proteins captured by the bait proteins are confined withinthe nano-clusters.

FIG. 13 is a schematic diagram illustrating conceptually the formationof nano-clusters by irradiating two different nanoparticles and a methodfor regulating molecular function of a particular protein therebyaccording to an embodiment of the present invention. In this case, thefirst nanoparticle is generated as a heterocomplex self-assembled by thefirst fusion protein including the first self-assembled protein and alight-induced heterodimerized protein, and the second fusion proteinincluding the first self-assembled protein and the bait protein, and thesecond nanoparticle is generated by self-assembly of the third fusionprotein including the second self-assembled protein and the partnerprotein interacting with the light-induced heterodimerized protein. Ifthe light-induced heterodimerized protein and the partner proteindisplayed on the surface of the first nanoparticle and the secondnanoparticle respectively interact each other by light irradiation and anano-cluster is generated, the target protein captured by the baitprotein is confined within the nano-cluster.

FIG. 14 is a schematic diagram illustrating conceptually the formationof nano-clusters by irradiating and a method for regulating molecularfunction of a particular protein thereby according to another embodimentof the present invention. In this case, the nanoparticle is ahomocomplex generated by self-assembly of a first fusion proteinincluding the self-assembled protein (SAP) and the light-inducedheterodimerized protein, and a nanocluster is generated when a lighthaving wavelength capable of inducing heterodimerization between thelight-induced heterodimerized protein and the partner protein afterco-expressing the first fusion protein and the second fusion proteinincluding the partner protein interacting with the light-inducedheterodimerized protein and a bait protein in a cell. As a result, thetarget protein which is an inherent protein in the cell is captured bythe bait protein and is confined within the nano-cluster thereby.

FIG. 15 is a schematic diagram illustrating conceptually the formationof nano-clusters by irradiating and a method for regulating molecularfunction of a particular protein thereby according to still anotherembodiment of the present invention. As shown in FIG. 15, thenanoparticle is a homocomplex generated by self-assembly of a firstfusion protein including a self-assembled protein and a light-inducedheterodimerized protein forming a homodimer regardless of lightirradiation (e.g., PHR), and if heterodimerization between thelight-induced heterodimerized protein and the partner protein is inducedby light irradiation after co-expressing the first fusion protein andthe second fusion protein including the partner protein interacting withthe light-induced heterodimerized protein and a bait protein in a cell.As a result, the target protein which is an inherent protein in the cellis captured by the bait protein and is confined within the nano-clusterthereby.

FIG. 16 is a schematic diagram illustrating conceptually the formationof nano-clusters by irradiating and the regulation of function of PI3Kprotein using the method illustrated in FIG. 14 according to anembodiment of the present invention.

FIG. 17 is a schematic diagram illustrating conceptually the formationof nano-clusters by irradiating according to an embodiment of thepresent invention and the regulation of function of Vav2 protein usingthe same. An intracellular protein, p110 (PI3K catalytic subunit)phosphorylates a substrate attached to cell membrane, PIP2(phosphatidyl-inositol-3,4-bisphosphate) when a growth factor such asPDGF (platelet-derived growth factor) is treated to a cell. If PIP3(phosphatidyl-inosito-3,4,5-triphosphate) is generated by thephosphorylation of PIP2, a PIP3 biosensor, PH domain of Akt proteinexisting in cytoplasm translocates to cell membrane by recognizing theresulting PIP3. However, the phosphorylation of PIP2 is inhibited andthe translocation of the PH domain to cell membrane may be inhibitedthereby if p110 is captured by iSH2 (inter Src homology 2 domain), aPI3K regulatory subunit as a bait protein by the photo-inducednano-cluster formation and confined within a nano-cluster thereby.

The present inventors confirmed experimentally that nanoclusters weregenerated due to the interaction between nanoparticles containing CIBN(N-terminal of cryptochrome-interacting basic-helix-loop-helix) forminga light-induced heterodimer with PHR (photolyase homologous region ofcryptochrome 2) and the PHR, and Vav2 proteins were captured within thenanoclusters and the formation of lamellipodia which is induced by theVav2 proteins was inhibited thereby, when a fusion protein containing anassociation domain (AD) of CaMKIIα involving in self-assembly and theCIBN, and a fusion protein containing the PHR and the Vav2 protein (atarget protein) were co-expressed in a cell and nanoparticles weregenerated by the self-assembly of the AD of CaMKIIα and blue light with488 nm of wavelength capable of forming the heterodimer between the CIBNand the PHR was irradiated (See FIG. 19). On the other hand, if a geneconstruct lacking a gene encoding the target protein (Vav2 protein) wasused, the nanoclusters were generated but the formation of thelamellipodia was not affected. (See FIG. 19). Therefore, the methods andkits according to the present invention are useful tools for regulatinga particular protein expressed or inherent in a cell and biochemicallyrelated downstream proteins effectively and in a reversible manner.

MODE FOR INVENTION Example 1: Construction of Vectors

1-1: Preparation of CIBN-CFP-AD Constructs

The present inventors constructed a polynucleotide encoding a fusionprotein in which CFP (cyan fluorescent protein) and an N-terminal (1-170a.a.) of CIB1 (cryptochrome-interacting basic-helix-loop-helix, GenBankNo.: NM_119618) is serially fused to N-terminal of AD (315-478 a.a.) ofCaMKIIα (GenBank No.: NM_012920) involving in self-assembly of theCaMIIα, and thus constructed resulting a CIBN-CFP-AD construct byinserting the polynucleotide into a pCFP-C1 vector replacing apolynucleotide encoding CFP of the vector with the polynucleotideencoding the fusion protein (FIG. 1.).

1-2: Preparation of PHR_(CRY2)-mCherry Construct

A polynucleotide encoding a polypeptide fragment (1-498 a.a.)corresponding PHR (phytolyase homologous region) domain of CYR2 protein(GenBank No. NM_100320) was inserted into pmCherry-N1 vector (Clontech,USA) and the resulting PHRCRY2-mCherry construct was prepared thereby.The CRY protein and the PHR form a heterodimer when expressed in a cell(FIG. 1).

Example 2: Preparation of Vectors for Analyzing Protein Interaction

The present inventors constructed expression vectors capable ofexpressing a bait protein and a target protein as described below, inorder to investigate whether the method for analyzing proteininteraction using the light-induced nanocluster formation may be usedfor analyzing the interaction between other proteins based on the resultof example 1.

2-1: Preparation of PHR-CCF)-Hras(Active) Construct

The present inventors constructed PHR-ECFP-Hras(active) construct byproducing a polynucleotide encoding a fusion protein in which the PHRdomain (1-498 a.a.) of CRY2 (GenBank No.: NM_100320) and a bait protein,active form Hras (GenBank No.: NM_001130442.1, Q61L mutant,CAAX-deleted) are fused to N-temmal and C-terminal of ECFP, respectivelyand inserting the polynucleotide into pECFP-C1 vector (Clontech, USA).

2-2: Preparation of YFP-RBD_(raf1) Construct

The present inventors constructed YFP-RBD_(raf1) construct by insertingoperably a polynucleotide encoding Ras binding domain (RBD_(raf1),51-131 a.a.) of Raf1 (GenBank No.; NM_002880.3) into a 3′-end of apolynucleotide encoding YFP of pYFP vector (Clontech, USA).

2-3: Preparation of YFP-RBD_(p110) Construct

The present inventors constructed YFP-RBD_(p110) construct by insertinga polynucleotide encoding Ras binding domain (RBD_(p110), 133-332 a.a.)of p110 alpha (GenBank No.: NM_006218.2) into a 3′-end of apolynucleotide encoding YFP of pYFP (Clontech, USA).

2-4: Preparation of PHR-ECFP-Hras(Inactive) Construct

PHR-ECFP-Hras(inactive) construct was constructed by substitutingHras(active) gene with Hras(inactive) gene (S17N mutant, CAAX-deleted)in the PHR-ECFP-Hras(active) construct prepared in example 2-1.

2-5: Preparation of YFP-CRIB_(RAK1) Construct

pYFP-CRIB_(PAK1) construct was constructed by substituting RBD_(p110)gene with CRIB_(PAK1) gene (GenBank No.: NM_001128620.1, 69-108 a.a.) inthe YFP-RBD_(p110) construct prepared in example 2-3.

Example 3: Construction of Vectors for Inhibiting a Protein Reversibly

3-1: Preparation of CIBN-mCerulean-AD Construct

CIBN-mCerulean-AD construct was constructed by inserting apolynucleotide encoding a fusion protein in which a cyan fluorescentprotein (mCerulean) and an N-terminal (1-170 a.a.) of CIB1 proteinparticipating in light-induced heterodimerizing are serially fused tothe N-terminal of activation domain (315-478 a.a.) into pmCerulean-C1vector (Clontech, USA) instead of the polynucleotide encoding themCerulean.

3-2: Preparation of mCitrine-PHR_(CRY2) Construct

mCitrine-PHR_(CRY2) construct was constructed by inserting apolynucleotide encoding a polypeptide corresponding to the PHR of CYR2protein (1-498 a.a. of CRY2) into pmCitrine-C1 vector (Clontech, USA).

3-3: Preparation of mCitrine-PHR_(CRY2)-Rit Tail Construct

mCitrine-PHR_(CYR2)-Rit tail construct was constructed by inserting apolynucleotide encoding a tail domain (193-219 a.a.) of Rit (GenBankNo.: NM_006912) which is localized to plasma membrane into themCitrine-PHR_(CYR2) construct whereby the Rit tail is fused to theC-terminal of PHR. The Rit tail is a protein capable of attachingelectrically to phospholipid molecules having negative charge andcapturing a protein in the plasma membrane.

3-4: Preparation of mCitrine-PHR_(CRY2)-DHPH_(Vav2)-Rit Tail Construct

mCitrine-PHR_(CRY2)-DHPH_(Vav2)-Rit tail construct was constructed byinserting a polynucleotide encoding a fusion protein in which apolypeptide corresponding a PHR domain (1-498 a.a.) of CRY2 (GenBankNo.: NM_100320), a DH-PH domain (167-541 a.a.) of Vav2 (GenBank No.:NM_001134398) inducing the formation of lamellipodia as a target proteinand a tail domain (193-219 a.a.) of Rit (GenBank No.: NM_006912) areserially fused to the C-terminal of the mCitrine into pmCitrine-C1vector (Clontech, USA). Since the Vav2 protein has PH domain and mayactivate proteins localized in plasma membrane by translocating intoplasma membrane weakly, it may perform recruiting of a protein intoplasma membrane. However, the present inventors used a Rit tailconstruct in order to recruit Vav2 protein into plasma membrane clearly.

3-5: Preparation of mCherry-Lifeact Construct

mCherry-lifeact construct was constructed by inserting a polyncleotideencoding lifeact, a marker for visualizing F-actin, which consists of anN-terminal fragment (1-17 a.a.) of Abp140 (actin binding protein 140,GenBank No.: NM_001183658) into pmCherry-C1 vector (Clontech, USA).

Experimental Example 1: Investigation of Light-Induced NanoclusterFormation

The present inventors observed intracellular fluorescent patterns ofCos-7 cells (ATCC no. CRL-1651) co-transfected with the CIBN-RFP-FTconstruct and the CRY2 construct prepared by the example 1 irradiatedwith blue light having 488 nm of wavelength using pulsed irradiation(for total 5 secs with 1 sec per irradiation at an interval of 12 secs).As a result, when the blue light was irradiated, red fluorescencedispersed within cytosol was displayed in a pattern including aplurality of strong red dots and then was dispersed in the cytosol againover time (FIG. 2). Although analyzing the phenomenon as the number ofstrong red dots, the number got increased when the blue light wasirradiated and got decreased gradually over time (FIG. 3). Therefore,the present inventors proved that a nanoparticle generated byself-assembly of a fusion protein including a light-inducedheterodimerized protein and a self-assembled protein may be clustered bythe interaction of the light-induced heterodimerized protein and apartner protein capable of heterodimerizing with the light-inducedheterodimerized protein by light irradiation and then be visualized byfluorescent proteins.

Experimental Example 2: Analysis of Protein-Protein Interaction

2-1: Co-Transfection of CIBN-RFP-AD, PHR-ECFP-Hras(Active) andYFP-RBD_(raf1) Constructs

The CIBN-RFP-AD construct prepared in the example 1-1, thePHR-CFP-Hras(active) construct prepared in the example 2-1 and theYFP-RBD_(raf1) construct prepared in the example 2-2 were co-transfectedinto Cos-7 cells (ATCC No. CRL-1651) using electroporation method (1,000V, 35 ms, 2 pulse) and fusion proteins were expressed by cultivating thetransected cells for 24 hour which were plated on 96-well glass bottomplate (Matrical Bioscience, USA) in a incubator under the condition of10% CO₂ and 37° C.

2-2: Co-Transfection of CIBN-RFP-AD, PHR-ECFP-Hras(Active) andYFP-RBD_(p110) Constructs

The CIBN-RFP-AD construct prepared in the example 1-1, thePHR-CFP-Hras(active) construct prepared in the example 2-1 and theYFP-RBD_(p110) construct prepared in the example 2-3 were co-transfectedinto Cos-7 cells (ATCC No. CRL-1651) using electroporation method (1,000V, 35 ms, 2 pulse) and fusion proteins were expressed by cultivating thetransfected cells for 24 hour which were plated on 96-well glass bottomplate (Matrical Bioscience, USA) in a incubator under the condition of10% CO₂ and 37° C.

2-3: Co-Transfection of CIBN-RFP-AD, PHR-ECFP-Hras(Inactive) andYFP-RBD_(raf1) Constructs

The CIBN-RFP-AD construct prepared in the example 1-1, thePHR-CFP-Hras(inactive) construct prepared in the example 2-4 as anegative control and the YFP-RBD_(raf1) construct prepared in theexample 2-2 were co-transfected into Cos-7 cells (ATCC No. CRL-1651)using electroporation method (1,000 V, 35 ms, 2 pulse) and fusionproteins were expressed by cultivating the transfected cells for 24 hourwhich were plated on 96-well glass bottom plate (Matrical Bioscience,USA) in a incubator under the condition of 10% CO₂ and 37° C.

2-4: Co-Transfection of CIBN-RFP-AD, PHR-ECFP-Hras(Active) andYFP-CRIB_(PAK1) Constructs

The CIBN-RFP-AD construct prepared in the example 1-1, thePHR-CFP-Hras(active) construct prepared in the example 2-1 and theYFP-CRIB_(PAK1) construct prepared in the example 2-5 as anothernegative control were co-transfected into Cos-7 cells (ATCC No.CRL-1651) using electroporation method (1,000 V. 35 ms, 2 pulse) andfusion proteins were expressed by cultivating the transfected cells for24 hour which were plated on 96-well glass bottom plate (MatricalBioscience, USA) in a incubator under the condition of 10% CO₂ and 37°C.

2-5: Analysis of Nanocluster Formation by Light Irradiation

The present inventors observed fluorescent patter within cellsco-transfected in the above experimental examples 2-1 to 2-4 afterirradiating blue light having 488 nm of wavelength capable of inducingthe interaction between CIBN and CRY2 using pulsed irradiation (fortotal 7 secs with 1 sec per irradiation at an interval of 10 secs) (FIG.18). As a result, when cells co-transfected with the CIBN-RFP-ADconstruct, the PHR-ECFP-Hras(active) construct and the YFP-RBDraf1construct were irradiated with blue light, patterns of red, cyan andyellow fluorescence shifted from dispersed ones to strong dotted onesand the locations of fluorescence for each color were same and thus itwas confirmed that the fluorescent proteins emitting each color wereco-located (FIG. 18a ). This means that nanoparticles generated by AD ofCalMKIIα, a self-assembled protein formed nano-clusters due tointermolecular interaction and it proves that the PHR-ECFP-Hra(active)fusion protein forming a homodimer interacted with the CIBN-RFP-ADfusion protein forming a self-assembled nanoparticle and Hras(active)which is used as a bait protein interacted with RBD_(raf1), a targetprotein.

In addition, when cells co-transfected with the CIBN-RFP-AD construct,PHR-ECFP-Hras(active) construct and the YFP-RBD_(p110) were irradiatedwith blue light, patterns of red, cyan and yellow fluorescence shiftedfrom dispersed one to strong dotted ones and the locations offluorescence for each color were same like the above and thus it wasconfirmed that the fluorescent proteins emitting each color wereco-located (FIG. 18c ).

On the other hand, when cells co-transfected with the CIBN-RFP-ADconstruct, the PHR-ECFP-Hras(inactive) construct and the YFP-RBD_(raf1)construct (FIG. 18b ) and cells co-transfected with the CIBN-RFP-ADconstruct, the PHR-ECFP-Hras(active) construct and the YFP-CRIB_(PAK1)construct (FIG. 18d ) were irradiated with blue light, patterns of redand cyan fluorescence shifted from dispersed one to strong dotted onesbut patterns of yellow fluorescence had no change.

This is interpreted if there is no interaction between the bait proteinand the target protein (FIG. 7). That is, nanoclusters are generatedwhen the CIBN, a light-induced heterodimerized protein interacts withPHR-ECFP-bait protein forming a homodimer by light irradiation but theinteraction between the bait protein and the target protein fails andthe target protein cannot form a complex with the nano-clusters and thusthe fluorescence emitted from the target protein is displayed in adispersed pattern.

In the meanwhile, if an homodimerization is occurred between thelight-induced heterodimerized protein, nano-clusters are generatedregardless of light irradiation and the interaction with the partnerprotein is inhibited, it can be distinguished from the interactionbetween the light-induced heterodimerized protein and the partnerprotein.

As such, the method and the kit for analyzing protein-proteininteraction according to various embodiments of the present inventionmay be useful in analyzing the interaction between different proteinsexcluding the possibility of false-positive and false-negative.

Experimental Example 3: Analysis of Protein Inhibition by LightIrradiation

The present inventors observed fluorescent patterns within Cos-7 cells(ATCC No. CRL-1658) co-transfected with the CIBN-mCerulean-Ad constructprepared in the example 3-1, the mCitrine-PHR_(CRY2)-DHPH_(Vav2)-Rittail construct prepared in the example 3-4 and the mCherry-lifeactconstruct prepared in the example 3-5 over time after irradiating bluelight having 488 nm of wavelength capable of inducing the interactionbetween CIBN and CRY2 using pulsed irradiation (for total 4 min with 1sec per irradiation at an interval of 10 secs). As a result, it wasconfirmed that nano-clusters were generated by light irradiation fromthe phenomenon that strong green fluorescent dots were generated and theformation of lamellipodia was inhibited (FIG. 19). The present inventorsperformed similar experiment using the mCitrine-PHR_(CRY2)-Rit tailconstruct prepared in the example 1-3 instead of themCitrine-PHR_(CRY2)-DHPH_(Vav2)-Rit tail construct prepared in example3-4 in order to investigate whether the inhibition of lamellipodiaformation is due to the capture of Vav2 protein within the nano-cluster.As a result, as shown in FIG. 19, nano-clusters were generated by lightirradiation, but it was irrelevant to the formation of lamellipodia.

While the present invention has been described in connection withcertain exemplary examples, it is to be understood that the invention isnot limited to the disclosed examples, but, on the contrary, is intendedto cover various modifications and equivalent arrangements includedwithin the spirit and scope of the appended claims, and equivalentsthereof.

INDUSTRIAL APPLICABILITY

The method for generating light-induced protein nano-clusters accordingto an embodiment of the present invention may be used as a useful toolfor analyzing various protein-protein interaction and analyzing functionof proteins by reversible inhibition of the proteins.

SEQUENCE LISTING FREE TEXT

SEQ ID NO: 1 is the amino acid sequence of the tetracysteine fluorescentmotif.

The invention claimed is:
 1. A method for forming a proteinnano-cluster, wherein the method comprises: providing a first expressionvector including a polynucleotide encoding a first fusion proteincomprising a light-induced heterodimerized protein and a firstself-assembled protein, and a second expression vector including apolynucleotide encoding a partner protein capable of forming aheterodimer with the light-induced heterodimerized protein, or a secondfusion protein containing the partner protein and a secondself-assembled protein; transforming a cell, a tissue or a subject withthe first expression vector and the second expression vector;irradiating light having wavelength capable of inducingheterodimerization between the light-induced heterodimerized protein andthe partner protein to the cell, the tissue or the subject; and forminga protein nano-cluster.
 2. The method according to claim 1, wherein thelight-induced heterodimerized protein is CIB, CIBN, PhyB, PIF, FKF1,GIGANTEA, CRY, or PHR, and the partner protein is CRY or PHR when thelight-induced heterodimerized protein is CIB or CIBN, the partnerprotein is PIF when the light-induced heterodimerized protein is PhyB,the partner protein is GIFANTEA when the light-induced heterodimerizedprotein is FKF1, the partner protein is CIB, CIBN, PhyB, PIF, FKF1,GIGANTEA, CRY, or PHR.
 3. The method according to claim 1, wherein thefirst self-assembled protein and the second self-assembled protein isindependently ferritin, virus capsid protein, ferritin-like protein,calcium/calmodulin-dependent protein kinase II alpha subunit (CaMKIIα)or DsRed.
 4. The method according to claim 1, wherein at least one amongthe first fusion protein, the partner protein and the second fusionprotein further comprises a fluorescent protein.
 5. The method accordingto claim 4, wherein the fluorescent protein is added to N-terminus orC-terminus of the first fusion protein, the partner protein and/or thesecond fusion protein or the fluorescent protein is inserted between thelight-induced heterodimerized protein and the first self-assembledprotein or between the partner protein and the second self-assembledprotein.
 6. The method according to claim 4, wherein the fluorescentprotein is green fluorescent protein (GFP), yellow fluorescent protein(YFP), red fluorescent protein (RFP), orange fluorescent protein, cyanfluorescent protein (CFP), blue fluorescent protein (BFP), ortetracysteine fluorescent motif.
 7. The method according to claim 5,wherein the fluorescent protein is green fluorescent protein (GFP),yellow fluorescent protein (YFP), red fluorescent protein (RFP), orangefluorescent protein, cyan fluorescent protein (CFP), blue fluorescentprotein (BFP), or tetracysteine fluorescent motif.